Methods and compositions for RNA interference

ABSTRACT

The present invention provides methods for attenuating gene expression in a cell using gene-targeted double stranded RNA (dsRNA). The dsRNA contains a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the gene to be inhibited (the “target” gene).

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of PCT applicationPCT/US01/08435, filed Mar. 16, 2001, and claims the benefit of U.S.Provisional applications U.S. Ser. No. 60/189,739 filed Mar. 16, 2000and U.S. Ser. No. 60/243,097 filed Oct. 24, 2000. The specifications ofsuch applications are incorporated by reference herein.

GOVERNMENT SUPPORT

[0002] Work described herein was supported by National Institutes ofHealth Grant R01-GM62534. The United States Government may have certainrights in the invention.

BACKGROUND OF THE INVENTION

[0003] “RNA interference”, “post-transcriptional gene silencing”,“quelling”—these different names describe similar effects that resultfrom the overexpression or misexpression of transgenes, or from thedeliberate introduction of double-stranded RNA into cells (reviewed inFire A (1999) Trends Genet 15:358-363; Sharp PA (1999) Genes Dev13:139-141; Hunter C (1999) Curr Biol 9:R440-R442; Baulcombe DC (1999)Curr Biol 9:R599-R601; Vaucheret et al. (1998) Plant J 16:651-659). Theinjection of double-stranded RNA into the nematode Caenorhabditiselegans, for example, acts systemically to cause thepost-transcriptional depletion of the homologous endogenous RNA (Fire etal. (1998) Nature 391: 806-811; and Montgomery et al. (1998) PNAS95:15502-15507). RNA interference, commonly referred to as RNAi, offersa way of specifically and potently inactivating a cloned gene, and isproving a powerful tool for investigating gene function. But thephenomenon is interesting in its own right; the mechanism has beenrather mysterious, but recent research—the latest reported by Smardon etal. (2000) Curr Biol 10:169-178—is beginning to shed light on the natureand evolution of the biological processes that underlie RNAi.

[0004] RNAi was discovered when researchers attempting to use theantisense RNA approach to inactivate a C. elegans gene found thatinjection of sense-strand RNA was actually as effective as the antisenseRNA at inhibiting gene function. Guo et al. (1995) Cell 81:611-620.Further investigation revealed that the active agent was modest amountsof double-stranded RNA that contaminate in vitro RNA preparations.Researchers quickly determined the ‘rules’ and effects of RNAi. Exonsequences are required, whereas introns and promoter sequences, whileineffective, do not appear to compromise RNAi (though there may begene-specific exceptions to this rule). RNAi acts systemically—injectioninto one tissue inhibits gene function in cells throughout the animal.The results of a variety of experiments, in C. elegans and otherorganisms, indicate that RNAi acts to destabilize cellular RNA after RNAprocessing.

[0005] The potency of RNAi inspired Timmons and Fire (1998 Nature 395:854) to do a simple experiment that produced an astonishing result. Theyfed to nematodes bacteria that had been engineered to expressdouble-stranded RNA corresponding to the C. elegans unc-22 gene.Amazingly, these nematodes developed a phenotype similar to that ofunc-22 mutants that was dependent on their food source. The ability toconditionally expose large numbers of nematodes to gene-specificdouble-stranded RNA formed the basis for a very powerful screen toselect for RNAi-defective C. elegans mutants and then to identify thecorresponding genes.

[0006] Double-stranded RNAs (dsRNAs) can provoke gene silencing innumerous in vivo contexts including Drosophila, Caenorhabditis elegans,planaria, hydra, trypanosomes, fungi and plants. However, the ability torecapitulate this phenomenon in higher eukaryotes, particularlymammalian cells, has not be accomplished in the art. Nor has the priorart demonstrated that this phenomena can be observe in culturedeukaryotes cells.

SUMMARY OF THE INVENTION

[0007] One aspect of the present invention provides a method forattenuating expression of a target gene in cultured cells, comprisingintroducing double stranded RNA (dsRNA) into the cells in an amountsufficient to attenuate expression of the target gene, wherein the dsRNAcomprises a nucleotide sequence that hybridizes under stringentconditions to a nucleotide sequence of the target gene.

[0008] Another aspect of the present invention provides a method forattenuating expression of a target gene in a mammalian cell, comprising

[0009] (i) activating one or both of a Dicer activity or an Argonautactivity in the cell, and

[0010] (ii) introducing into the cell a double stranded RNA (dsRNA) inan amount sufficient to attenuate expression of the target gene, whereinthe dsRNA comprises a nucleotide sequence that hybridizes understringent conditions to a nucleotide sequence of the target gene.

[0011] In certain embodiments, the cell is suspended in culture; whilein other embodiments the cell is in a whole animal, such as a non-humanmammal.

[0012] In certain preferred embodiments, the cell is engineered with (i)a recombinant gene encoding a Dicer activity, (ii) a recombinant geneencoding an Argonaut activity, or (iii) both. For instance, therecombinant gene may encode, for a example, a protein which includes anamino acid sequence at least 50 percent identical to SEQ ID No. 2 or 4;or be defined by a coding sequence hybridizes under wash conditions of2×SSC at 22° C. to SEQ ID No. 1 or 3. In certain embodiments, therecombinant gene may encode, for a example, a protein which includes anamino acid sequence at least 50 percent identical to the Argonautsequence shown in FIG. 24.

[0013] In certain embodiments, rather than use a heterologous expressionconstruct(s), an endogenous Dicer gene or Argonaut gene can beactivated, e.g, by gene activation technology, expression of activatedtranscription factors or other signal transduction protein, whichinduces expression of the gene, or by treatment with an endogenousfactor which upregualtes the level of expression of the protein orinhibits the degradation of the protein.

[0014] In certain preferred embodiments, the target gene is anendogenous gene of the cell. In other embodiments, the target gene is anheterologous gene relative to the genome of the cell, such as a pathogengene, e.g., a viral gene.

[0015] In certain embodiments, the cell is treated with an agent thatinhibits protein kinase RNA-activated (PKR) apoptosis, such as bytreatment with agents which inhibit expression of PKR, cause itsdestruction, and/or inhibit the kinase activity of PKF.

[0016] In certain preferred embodiments, the cell is a primate cell,such as a human cell.

[0017] In certain preferred embodiments, the length of the dsRNA is atleast 20, 21 or 22 nucleotides in length, e.g., corresponding in size toRNA products produced by Dicer-dependent cleavage. In certainembodiments, the dsRNA construct is at least 25, 50, 100, 200, 300 or400 bases. In certain embodiments, the dsRNA construct is 400-800 basesin length.

[0018] In certain preferred embodiments, expression of the target geneis attenuated by at least 5 fold, and more preferably at least 10, 20 oreven 50 fold, e.g., relative to the untreated cell or a cell treatedwith a dsRNA construct which does not correspond to the target gene.

[0019] Yet another aspect of the present invention provides a method forattenuating expression of a target gene in cultured cells, comprisingintroducing an expression vector having a “coding sequence” which, whentranscribed, produces double stranded RNA (dsRNA) the cell in an amountsufficient to attenuate expression of the target gene, wherein the dsRNAcomprises a nucleotide sequence that hybridizes under stringentconditions to a nucleotide sequence of the target gene. An certainembodiments, the vector includes a single coding sequence for the dsRNAwhich is operably linked to (two) transcriptional regulatory sequenceswhich cause transcription of in both directions (to form complementarytranscripts of the coding sequence. In other embodiments, the vectorincludes two coding sequences which, respectively, give rise to the twocomplementary sequences which form the dsRNA when annealed. In certainembodiments, the vectors are episomal, e.g., and transfection istransient. In other embodiments, the vectors are chromosomallyintegrated, e.g., to produce a stably transfected cell line. Preferredvectors for forming such stable cell lines are the described in U.S.Pat. No. 6,025,192 and PCT publication WO/9812339, which areincorporated by reference herein.

[0020] Still another aspect of the present invention provides an assayfor identifying nucleic acid sequences responsible for conferring aparticular phenotype in a cell, comprising

[0021] (i) constructing a variegated library of nucleic acid sequencesfrom a cell in an orientation relative to a promoter to produce doublestranded DNA;

[0022] (ii) introducing the variegated dsRNA library into a culture oftarget cells, which cells have an activated Dicer activity or Argonautactivity;

[0023] (iii) identifying members of the library which confer aparticular phenotype on the cell, and identifying the sequence from acell which correspond, such as being identical or homologous, to thelibrary member.

[0024] Yet another aspect of the present invention provides a method ofconducting a drug discovery business comprising:

[0025] (i) identifying, by the subject assay, a target gene whichprovides a phenotypically desirable response when inhibited by RNAi;

[0026] (ii) identifying agents by their ability to inhibit expression ofthe target gene or the activity of an expression product of the targetgene;

[0027] (iii) conducting therapeutic profiling of agents identified instep (b), or further analogs thereof, for efficacy and toxicity inanimals; and

[0028] (iv) formulating a pharmaceutical preparation including one ormore agents identified in step (iii) as having an acceptable therapeuticprofile.

[0029] The method may include an additional step of establishing adistribution system for distributing the pharmaceutical preparation forsale, and may optionally include establishing a sales group formarketing the pharmaceutical preparation.

[0030] Another aspect of the present invention provides a method ofconducting a target discovery business comprising:

[0031] (i) identifying, by the subject assay, a target gene whichprovides a phenotypically desirable response when inhibited by RNAi;

[0032] (ii) (optionally) conducting therapeutic profiling of the targetgene for efficacy and toxicity in animals; and

[0033] (iii). licensing, to a third party, the rights for further drugdevelopment of inhibitors of the target gene.

[0034] Another aspect of the invention provides a method for inhibitingRNAi by inhibiting the expression or activity of an RNAi enzyme. Thus,the subject method may include inhibiting the acitivity of Dicer and/orthe 22-mer RNA.

[0035] Still another aspect relates to the a method for altering thespecificity of an RNAi by modifying the sequence of the RNA component ofthe RNAi enzyme.

[0036] Another aspect of the invention relates to purified orsemi-purified preparations of the RNAi enzyme or components thereof. Incertain embodiments, the preparations are used for identifyingcompounds, especially small organic molecules, which inhibit orpotentiate the RNAi activity. Small molecule inhibitors, for example,can be used to inhibit dsRNA responses in cells which are purposefullybeing transfected with a virus which produces double stranded RNA.

[0037] The dsRNA construct may comprise one or more strands ofpolymerized ribonucleotide. It may include modifications to either thephosphate-sugar backbone or the nucleoside. The double-strandedstructure may be formed by a single self-complementary RNA strand or twocomplementary RNA strands. RNA duplex formation may be initiated eitherinside or outside the cell. The dsRNA construct may be introduced in anamount which allows delivery of at least one copy per cell. Higher dosesof double-stranded material may yield more effective inhibition.Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition. dsRNA constructs containing a nucleotide sequences identicalto a portion of the target gene is preferred for inhibition. RNAsequences with insertions, deletions, and single point mutationsrelative to the target sequence have also been found to be effective forinhibition. Thus, sequence identity may optimized by alignmentalgorithms known in the art and calculating the percent differencebetween the nucleotide sequences. Alternatively, the duplex region ofthe RNA may be defined functionally as a nucleotide sequence that iscapable of hybridizing with a portion of the target gene transcript.

[0038] Yet another aspect of the invention pertains to transgenicnon-human mammals which include a transgene encoding a dsRNA construct,preferably which is stably integrated into the genome of cells in whichit occurs. The animals can be derived by oocyte microinjection, forexample, in which case all of the nucleated cells of the animal willinclude the transgene, or can be derived using embryonic stem (ES) cellswhich have been transfected with the transgene, in which case the animalis a chimera and only a portion of its nucleated cells will include thetransgene. In certain instances, the sequence-independent dsRNAresponse, e.g., the PKR response, is also inhibited in those cellsincluding the transgene.

[0039] In still other embodiments, dsRNA itself can be introduced intoan ES cell in order to effect gene silencing, and that phenotype will becarried for at least several rounds of division, e.g., into the progenyof that cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040]FIG. 1: RNAi in S2 cells. a, Drosophila S2 cells were transfectedwith a plasmid that directs lacZ expression from the copia promoter incombination with dsRNAs corresponding to either human CD8 or lacZ, orwith no dsRNA, as indicated. b, S2 cells were co-transfected with aplasmid that directs expression of a GFP-US9 fusion protein (12) anddsRNAs of either lacZ or cyclin E, as indicated. Upper panels show FACSprofiles of the bulk population. Lower panels show FACS profiles fromGFP-positive cells. c, Total RNA was extracted from cells transfectedwith lacZ, cyclin E, fizzy or cyclin A dsRNAs, as indicated. Northernblots were hybridized with sequences not present in the transfecteddsRNAs.

[0041]FIG. 2: RNAi in vitro. a, Transcripts corresponding to either thefirst 600 nucleotides of Drosophila cyclin E (E600) or the first 800nucleotides of lacZ (Z800) were incubated in lysates derived from cellsthat had been transfected with either lacZ or cyclin E (cycE) dsRNAs, asindicated. Time points were 0, 10, 20, 30, 40 and 60 min for cyclin Eand 0, 10, 20, 30 and 60 min for lacZ. b, Transcripts were incubated inan extract of S2 cells that had been transfected with cyclin E dsRNA(cross-hatched box, below). Transcripts corresponded to the first 800nucleotides of lacZ or the first 600, 300, 220 or 100 nucleotides ofcyclin E, as indicated. Eout is a transcript derived from the portion ofthe cyclin E cDNA not contained within the transfected dsRNA. E-ds isidentical to the dsRNA that had been transfected into S2 cells. Timepoints were 0 and 30 min. c, Synthetic transcripts complementary to thecomplete cyclin E cDNA (Eas) or the final 600 nucleotides (Eas600) or300 nucleotides (Eas300) were incubated in extract for 0 or 30 min.

[0042]FIG. 3: Substrate requirements of the RISC. Extracts were preparedfrom cells transfected with cyclin E dsRNA. Aliquots were incubated for30 min at 30° C. before the addition of either the cyclin E (E600) orlacZ (Z800) substrate. Individual 20-μl aliquots, as indicated, werepre-incubated with 1 mM CaCl₂ and 5 mM EGTA, 1 mM CaCl₂, 5 mM EGTA and60 U of micrococcal nuclease, 1 mM CaCl₂ and 60 U of micrococcalnuclease or 10 U of DNase I (Promega) and 5 mM EGTA. After the 30-minpre-incubation, EGTA was added to those samples that lacked it. YeasttRNA (1 μg) was added to all samples. Time points were at 0 and 30 min.

[0043]FIG. 4: The RISC contains a potential guide RNA. a, Northern blotsof RNA from either a crude lysate or the S100 fraction (containing thesoluble nuclease activity, see Methods) were hybridized to a riboprobederived from the sense strand of the cyclin E mRNA. b, Solublecyclin-E-specific nuclease activity was fractionated as described inMethods. Fractions from the anion-exchange resin were incubated with thelacZ, control substrate (upper panel) or the cyclin E substrate (centrepanel). Lower panel, RNA from each fraction was analysed by northernblotting with a uniformly labelled transcript derived from sense strandof the cyclin E cDNA. DNA oligonucleotides were used as size markers.

[0044]FIG. 5: Generation of 22 mers and degradation of mRNA are carriedout by distinct enzymatic complexes. A. Extracts prepared either from0-12 hour Drosophila embryos or Drosophila S2 cells (see Methods) wereincubated 0, 15, 30, or 60 minutes (left to right) with auniformly-labeled double-stranded RNA corresponding to the first 500nucleotides of the Drosophila cyclin E coding region. M indicates amarker prepared by in vitro transcription of a synthetic template. Thetemplate was designed to yield a 22 nucleotide transcript. The doubletmost probably results from improper initiation at the +1 position. B.Whole-cell extracts were prepared from S2 cells that had beentransfected with a dsRNA corresponding to the first 500 nt. of theluciferase coding region. S10 extracts were spun at 30,000×g for 20minutes which represents our standard RISC extract⁶. S100 extracts wereprepared by further centrifugation of S10 extracts for 60 minutes at100,000×g. Assays for mRNA degradation were carried out as describedpreviously⁶ for 0, 30 or 60 minutes (left to right in each set) witheither a single-stranded luciferase mRNA or a single-stranded cyclin EmRNA, as indicated. C. S10 or S100 extracts were incubated with cyclin EdsRNAs for 0, 60 or 120 minutes (L to R).

[0045]FIG. 6: Production of 22 mers by recombinant CG4792/Dicer. A.Drosophila S2 cells were transfected with plasmids that direct theexpression of T7-epitope tagged versions of Drosha, CG4792/Dicer-1 andHomeless. Tagged proteins were purified from cell lysates byimmunoprecipitation and were incubated with cyclin E dsRNA. Forcomparison, reactions were also performed in Drosophila embryo and S2cell extracts. As a negative control, immunoprecipitates were preparedfrom cells transfected with a β-galactosidase expression vector. Pairsof lanes show reactions performed for 0 or 60 minutes. The syntheticmarker (M) is as described in the legend to FIG. 1. B. Diagrammaticrepresentations of the domain structures of CG4792/Dicer-1, Drosha andHomeless are shown. C. Immunoprecipitates were prepared from detergentlysates of S2 cells using an antiserum raised against the C-terminal 8amino acids of Drosophila Dicer-1 (CG4792). As controls, similarpreparations were made with a pre-immune serum and with an immune serumthat had been pre-incubated with an excess of antigenic peptide.Cleavage reactions in which each of these precipitates was incubatedwith an ˜500 nt. fragment of Drosophila cyclin E are shown. Forcomparsion, an incubation of the substrate in Drosophila embryo extractwas electrophoresed in parallel. D. Dicer immunoprecipitates wereincubated with dsRNA substrates in the presence or absence of ATP. Forcomparison, the same substrate was incubated with S2 extracts thateither contained added ATP or that were depleted of ATP using glucoseand hexokinase (see methods). E. Drosophila S2 cells were transfectedwith uniformly, 32P-labelled dsRNA corresponding to the first 500 nt. ofGFP. RISC complex was affinity purified using a histidine-tagged versionof D.m. Ago-2, a recently identified component of the RISC complex(Hammond et al., in prep). RISC was isolated either under conditions inwhich it remains ribosome associated (ls, low salt) or under conditionsthat extract it from the ribosome in a soluble form (hs, high salt)⁶.For comparison, the spectrum of labelled RNAs in the total lysate isshown. F. Guide RNAs produced by incubation of dsRNA with a Dicerimmunoprecipitate are compared to guide RNAs present in aaffinity-purified RISC complex. These precisely comigrate on a gel thathas single-nucleotide resolution. The lane labelled control is anaffinity selection for RISC from cell that had been transfected withlabeled dsRNA but not with the epitope-tagged D.m. Ago-2.

[0046]FIG. 7: Dicer participates in RNAi. A. Drosophila S2 cells weretransfected with dsRNAs corresponding to the two Drosophila Dicers(CG4792 and CG6493) or with a control dsRNA corresponding to murinecaspase 9. Cytoplasmic extracts of these cells were tested for Diceractivity. Transfection with Dicer dsRNA reduced activity in lysates by7.4-fold. B. The Dicer-1 antiserum (CG4792) was used to prepareimmunoprecipitates from S2 cells that had been treated as describedabove. Dicer dsRNA reduced the activity of Dicer-1 in this assay by6.2-fold. C. Cells that had been transfected two days previously witheither mouse caspase 9 dsRNA or with Dicer dsRNA were cotransfected witha GFP expression plasmid and either control, luciferase dsRNA or GFPdsRNA. Three independent experiments were quantified by FACS. Acomparison of the relative percentage of GFP-positive cells is shown forcontrol (GFP plasmid plus luciferase dsRNA) or silenced (GFP plamsidplus GFP dsRNA) populations in cells that had previously beentransfected with either control (caspase 9) or Dicer dsRNAs.

[0047]FIG. 8: Dicer is an evolutionarily conserved ribonuclease. A. Amodel for production of 22 mers by Dicer. Based upon the proposedmechanism of action of Ribonuclease III, we propose that Dicer acts onits substrate as a dimer. The positioning of the two ribonucleasedomains (RIIIa and RIIIb) within the enzyme would thus determine thesize of the cleavage product. An equally plausible alternative modelcould be derived in which the RIIIa and RIIIb domains of each Dicerenzyme would cleave in concert at a single position. In this model, thesize of the cleavage product would be determined by interaction betweentwo neighboring Dicer enzymes. B. Comparison of the domain structures ofpotential Dicer homologs in various organisms (Drosophila—CG4792,CG6493, C. elegans—K12H4.8, Arabidopsis—CARPEL FACTORY²⁴, T25K16.4,AC012328_(—)1, human Helicase-MOI²⁵ and S. pombe—YC9A_SCHPO). The ZAPdomains were identified both by analysis of individual sequences withPfam²⁷ and by Psi-blast²⁸ searches. The ZAP domain in the putative S.pombe Dicer is not detected by PFAM but is identified by Psi-Blast andis thus shown in a different color. For comparison, a domain structureof the RDE1/QDE2/ARGONAUTE family is shown. It should be noted that theZAP domains are more similar within each of the Dicer and ARGONAUTEfamilies than they are between the two groups. C. An alignment of theZAP domains in selected Dicer and Argonaute family members is shown. Thealignment was produced using ClustalW.

[0048]FIG. 9: Purification strategy for RISC. (second step in RNAimodel).

[0049]FIG. 10: Fractionation of RISC activity over sizing column.Activity fractionates as 500 KD complex. Also, antibody to dm argonaute2 cofractionates with activity.

[0050] FIGS. 11-13: Fractionation of RISC over monoS, monoQ,Hydroxyapatite columns. Dm argonaute 2 protein also cofactionates.

[0051]FIG. 14: Alignment of dm argonaute 2 with other family members.

[0052]FIG. 15: Confirmation of dm argonaute 2. S2 cells were transfectedwith labeled dsRNA and His tagged argonaute. Argonaute was isolated onnickel agarose and RNA component was identified on 15% acrylamide gel.

[0053]FIG. 16: S2 cell and embryo extracts were assayed for 22 mergenerating activity.

[0054]FIG. 17: RISC can be separated from 22 mer generating activity(dicer). Spinning extracts (S100) can clear RISC activity fromsupernatant (left panel) however, S100 spins still contain diceractivity (right panel).

[0055]FIG. 18: Dicer is specific for dsRNA and prefers longersubstrates.

[0056]FIG. 19: Dicer was fractionated over several columns.

[0057]FIG. 20: Identification of dicer as enzyme which can process dsRNAinto 22 mers. Various RNaseIII family members were expressed with nterminal tags, immunoprecipitated, and assayed for 22 mer generatingactivity (left panel). In right panel, antibodies to dicer could alsoprecipitate 22 mer generating activity.

[0058]FIG. 21: Dicer requires ATP.

[0059]FIG. 22: Dicer produces RNAs that are the same size as RNAspresent in RISC.

[0060]FIG. 23: Human dicer homolog when expressed and immunoprecipitatedhas 22 mer generating activity.

[0061]FIG. 24: Sequence of dm argonaute 2. Peptides identified bymicrosequencing are shown in underline.

[0062]FIG. 25: Molecular charaterization of dm argonaute 2. The presenceof an intron in coding sequence was determined by northern blottingusing intron probe. This results in a different 5′ reading frame thatthat published genome seqeunce. Number of polyglutaine repeats wasdetermined by genomic PCR.

[0063]FIG. 26: Dicer activity can be created in human cells byexpression of human dicer gene. Host cell was 293. Crude extracts haddicer activity, while activity was absent from untransfected cells.Activity is not dissimilar to that seen in drosophila embryo extracts.

[0064]FIG. 27: An ˜500 nt. fragment of the gene that is to be silenced(X) is inserted into the modified vector as a stable direct repeat usingstandard cloning procedures. Treatment with commercially available crerecombinase reverses sequences within the loxP sites (L) to create aninverted repeat. This can be stably maintained and amplified in an sbcmutant bacterial strain (DL759). Transcription in vivo from the promoterof choice (P) yields a hairpin RNA that causes silencing. A zeocinresistance marker is included to insure maintenance of the direct andinverted repeat structures; however this is non-essential in vivo andcould be removed by pre-mRNA splicing if desired. Smith, N. A. et al.Total silencing by intron-spliced hairpin RNAs. Nature 407, 319-20(2000).

[0065]FIG. 28: Hela, Chinese hamster ovary, and P19 (pluripotent, mouseembryonic carcinoma) cell lines transfected with plasmids expressingPhotinus pyralis (firefly) Renilla reniformis (sea pansy) luciferasesand with dsRNA 500 mers (400 ng), either homologous to fireflyluciferase mRNA (dsLUC) or non-homologous (dsGFP). Dual luciferaseassays were carried out using an Analytical Scientific Instruments model3010 Luminometer. In this assay Renilla luciferase serves as an internalcontrol for dsRNA-specific suppression of firefly luciferase activity.These data demonstrate that 500 mer dsRNA can specifically suppresscognate gene expression in vivo.

[0066]FIG. 29: P19 (a pluripontent, mouse embryonic cell line) cellstransfected with plasmids expressing Photinus pyralis (firefly) Renillareniformis (sea pansy) luciferases and with dsRNA 500 mers (500ng),either homologous to firefly luciferase mRNA (dsLUC) or non-homologous(dsGFP). Dual luciferase assays were carried out using an AnalyticalScientific Instruments model 3010 Luminometer. In this assay Renillaluciferase serves as an internal control for dsRNA-specific suppressionof firefly luciferase activity. These data further demonstrate that 500mer dsRNA can specifically suppress cognate gene expression in vivo andthat the effect is stable over time .

[0067]FIG. 30: S10 fractions from P19 cell lysates were used for invitro translations of mRNA coding for Photinus pyralis (firefly) Renillareniformis (sea pansy) luciferases. Translation reactions wereprogrammed with various amounts of dsRNA 500 mers, either homologous tofirefly luciferase mRNA (dsLUC) or non-homologous (dsGFP). Reactionswere carried out at 30 degrees for 1 hour, after which dual luciferaseassays were carried out using an Analytical Scientific Instruments model3010 Luminometer. In this assay Renilla luciferase serves as an internalcontrol for dsRNA-specific suppression of firefly luciferase activity.These data demonstrate that 500 mer dsRNA can specifically suppresscognate gene expression in vitro in a manner consistent withpost-transcriptional gene silencing. Anti-sense firefly RNA did notdiffer significantly from dsGFP control (approximately 10%) (data notshown).

[0068]FIG. 31: S10 fractions from P19 cell lysates were used for invitro translations of mRNA coding for Photinus pyralis (firefly) Renillareniformis (sea pansy) luciferases. Translation reactions wereprogrammed with dsRNA or asRNA 500 mers, either complementary to fireflyluciferase mRNA (asLUC and dsLUC) or non-complementary (dsGFP).Reactions were carried out at 30 degrees for 1 hour, after a 30 minpreincubation with dsRNA or asRNA. Dual luciferase assays were carriedout using an Analytical Scientific Instruments model 3010 Luminometer.In this assay Renilla luciferase serves as an internal control fordsRNA-specific suppression of firefly luciferase activity. These datademonstrate that 500 mer double-stranded RNA (dsRNA) but not anti-senseRNA (asRNA) suppresses cognate gene expression in vitro in a mannerconsistent with post-transcriptional gene silencing.

[0069]FIG. 32: P19 cells were grown in 6-well tissue culture plates toapproximately 60% confluence. Various amounts of dsRNA, eitherhomologous to firefly luciferase mRNA (dsLUC) or non-homologous (dsGFP),were added to each well and incubated for 12 hrs under normal tissueculture conditions. Cells were then transfected with plasmids expressingPhotinus pyralis (firefly) Renilla reniformis (sea pansy) luciferasesand with dsRNA 500 mers (500 ng). Dual luciferase assays were carriedout 12 hrs post-transfection using an Analytical Scientific Instrumentsmodel 3010 Luminometer. In this assay Renilla luciferase serves as aninternal control for dsRNA-specific suppression of firefly luciferaseactivity. These data show that 500 mer dsRNA can specifically suppresscognate gene expression in vivo without transfection under normal tissueculture conditions.

[0070]FIG. 33: Is a graph illustrating the relative rate of expressionluciferase in cells which are treated with various antisense and dsRNAconstructs.

DETAILED DESCRIPTION OF THE CERTAIN PREFERRED EMBODIMENTS I. Overview

[0071] The present invention provides methods for attenuating geneexpression in a cell using gene-targeted double stranded RNA (dsRNA).The dsRNA contains a nucleotide sequence that hybridizes underphysiologic conditions of the cell to the nucleotide sequence of atleast a portion of the gene to be inhibited (the “target” gene).

[0072] A significant aspect to certain embodiments of the presentinvention relates to the demonstration in the present application thatRNAi can in fact be accomplished in cultured cells, rather than wholeorganisms as described in the art.

[0073] Another salient feature of the present invention concerns theability to carry out RNAi in higher eukaryotes, particularly innon-oocytic cells of mammals, e.g., cells from adult mammals as anexample.

[0074] As described in further detail below, the present invention(s)are based on the discovery that the RNAi phenomenum is mediated by a setof enzyme activities, including an essential RNA component, that areevolutionarily conserved in eukaryotes ranging from plants to mammals.

[0075] One enzyme contains an essential RNA component. After partialpurification, a multi-component nuclease (herein “RISC nuclease”)co-fractionates with a discrete, 22-nucleotide RNA species which mayconfer specificity to the nuclease through homology to the substratemRNAs. The short RNA molecules are generated by a processing reactionfrom the longer input dsRNA. Without wishing to be bound by anyparticular theory, these 22 mer guide RNAs may serve as guide sequencesthat instruct the RISC nuclease to destroy specific mRNAs correspondingto the dsRNA sequences.

[0076] As illustrated in FIG. 33, double stranded forms of the 22-merguide RNA can be sufficient in length to induce sequence-dependent dsRNAinhibition of gene expression. In the illustrated example, dsRNAcontructs are administered to cells having a recombinant luciferasereporter gene. The control cell, e.g., no exogeneously added RNA, thelevel of expression of the luciferase reporter is normalized to be thevalue of “1”. As illustrated, both long (500-mer) and short (22-mer)dsRNA constructs complementary to the luciferase gene could inhibitexpression of that gene product relative to the control cell. On theother hand, similarly sized dsRNA complementary to the coding sequencefor another protein, green fluorescence protein (GFP), did notsignificantly effect the expression of luciferase—indicating that theinhibitory phenomena was in each case sequence-dependent. Likewise,single stranded 22-mers of luciferase did not inhibit expression of thatgene—indicating that the inhibitory phenomena is doublestranded-dependent.

[0077] The appended examples also identify an enzyme, Dicer, that canproduce the putative guide RNAs. Dicer is a member of the RNAse IIIfamily of nucleases that specifically cleave dsRNA and is evolutionarilyconserved in worms, flies, plants, fungi and, as described herein,mammals. The enzyme has a distinctive structure which includes ahelicase domain and dual RNAse III motifs. Dicer also contains a regionof homology to the RDE1/QDE2/ARGONAUTE family, which have beengenetically linked to RNAi in lower eukaryotes. Indeed, activation of,or overexpression of Dicer may be sufficient in many cases to permit RNAinterference in otherwise non-receptive cells, such as culturedeukaryotic cells, or mammalian (non-oocytic) cells in culture or inwhole organisms.

[0078] In certain embodiments, the cells can be treated with an agent(s)that inhibits the general double-stranded RNA response(s) by the hostcells, such as may give rise to sequence-independent apoptosis. Forinstance, the cells can be treated with agents that inhibit thedsRNA-dependent protein kinase known as PKR (protein kinaseRNA-activated). Double stranded RNAs in mammalian cells typicallyactivate protein kinase PKR and leads to apoptosis. The mechanism ofaction of PKR includes phosphorylation and inactivation eIF2a (Fire(1999) Trends Genet 15:358). It has also been reported that induction ofNF-κB by PKR is involved in apoptosis commitment and this process ismediated through activation of the IKK complex. Thissequence-independent response may reflect a form of primitive immuneresponse, since the presence of dsRNA is a common feature of many virallifecycles.

[0079] As described herein, Applicants have demonstrated that the PKRresponse can be overcome in favor of the sequence-specific RNAiresponse. However, in certain instances, it can be desirable to treatthe cells with agents which inhibit expression of PKR, cause itsdestruction, and/or inhibit the kinase activity of PKF are specificallycontemplated for use in the present method. Likewise, overexpression ofor agents which ectopic activate IF2αa. can be used. Other agents whichcan be used to suppress the PKR response include inhibitors of IKKphosphorylation of IκB, inhibitors of IκB ubiquitination, inhibitors ofIκB degradation, inhibitors of NF-κB nuclear translocation, andinhibitors of NF-κB interaction with κB response elements.

[0080] Other inhibitors of sequence-independent dsRNA response in cellsinclude the gene product of the vaccinia virus E3L. The E3L gene productcontains two distinct domains. A conserved carboxy-terminal domain hasbeen shown to bind double-stranded RNA (dsRNA) and inhibit the antiviraldsRNA response by cells. Expression of at least that portion of the E3Lgene in the host cell, or the use of polypeptide or peptidomimeticsthereof, can be used to suppress the general dsRNA response. Caspaseinhibitors sensitized cells to killing by double-stranded RNA.Accordingly, ectopic expression or activated of caspases in the hostcell can be used to suppress the general dsRNA response.

[0081] In other embodiments, the subject method is carried out in cellswhich have little or no general response to double stranded RNA, e.g.,have no PKR-dependent dsRNA response, at least under the cultureconditions. As illustrated in FIGS. 28-32, CHO and P19 cells can be usedwithout having to inhibit PKR or other general dsRNA responses.

[0082] Thus, the present invention provides a process and compositionsfor inhibiting expression of a target gene in a cell, expecially amammalian cell. In certain embodiments, the process comprisesintroduction of RNA (the “dsRNA construct”) with partial or fullydouble-stranded character into the cell or into the extracellularenvironment. Inhibition is specific in that a nucleotide sequence from aportion of the target gene is chosen to produce the dsRNA construct. Inpreferred embodiments, the method utilizes a cell in which Dicer and/orArgonaute activities are recombinantly expressed or otherwiseectopically activated. This process can be (1) effective in attenuatinggene expression, (2) specific to the targeted gene, and (3) general inallowing inhibition of many different types of target gene.

II. Definitions

[0083] For convenience, certain terms employed in the specification,examples, and appended claims are collected here.

[0084] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to that it hasbeen linked. One type of vector is a genomic integrated vector, or“integrated vector”, which can become integrated into the chromsomal DNAof the host cell. Another type of vector is an episomal vector, i.e., anucleic acid capable of extra-chromosomal replication. Vectors capableof directing the expression of genes to that they are operatively linkedare referred to herein as “expression vectors”. In the presentspecification, “plasmid” and “vector” are used interchangeably unlessotherwise clear from the context.

[0085] As used herein, the term “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, as applicableto the embodiment being described, single-stranded (such as sense orantisense) and double-stranded polynucleotides.

[0086] As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding a polypeptide ofthe present invention, including both exon and (optionally) intronsequences. A “recombinant gene” refers to nucleic acid encoding suchregulatory polypeptides, that may optionally include intron sequencesthat are derived from chromosomal DNA. The term “intron” refers to a DNAsequence present in a given gene that is not translated into protein andis generally found between exons. As used herein, the term“transfection” means the introduction of a nucleic acid, e.g., anexpression vector, into a recipient cell by nucleic acid-mediated genetransfer.

[0087] A “protein coding sequence” or a sequence that “encodes” aparticular polypeptide or peptide, is a nucleic acid sequence that istranscribed (in the case of DNA) and is translated (in the case of mRNA)into a polypeptide in vitro or in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, cDNA from procaryotic or eukaryoticmRNA, genomic DNA sequences from procaryotic or eukaryotic DNA, and evensynthetic DNA sequences. A transcription termination sequence willusually be located 3′ to the coding sequence.

[0088] Likewise, “encodes”, unless evident from its context, will bemeant to include DNA sequences that encode a polypeptide, as the term istypically used, as well as DNA sequences that are transcribed intoinhibitory antisense molecules.

[0089] The term “loss-of-function”, as it refers to genes inhibited bythe subject RNAi method, refers a diminishment in the level ofexpression of a gene when compared to the level in the absense of dsRNAconstructs.

[0090] The term “expression” with respect to a gene sequence refers totranscription of the gene and, as appropriate, translation of theresulting mRNA transcript to a protein. Thus, as will be clear from thecontext, expression of a protein coding sequence results fromtranscription and translation of the coding sequence.

[0091] “Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0092] The term “cultured cells” refers to cells suspended in culture,e.g., dispersed in culture or in the form tissue. It does not, however,include oocytes or whole embryos (including blastocysts and the like)which may be provided in culture. In certain embodiments, the culturedcells are adults cells, e.g., non-embryonic.

[0093] By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

[0094] As used herein, the terms “transduction” and “transfection” areart recognized and mean the introduction of a nucleic acid, e.g., anexpression vector, into a recipient cell by nucleic acid-mediated genetransfer. “Transformation”, as used herein, refers to a process in whicha cell's genotype is changed as a result of the cellular uptake ofexogenous DNA or RNA, and, for example, the transformed cell expresses adsRNA contruct.

[0095] “Transient transfection” refers to cases where exogenous DNA doesnot integrate into the genome of a transfected cell, e.g., whereepisomal DNA is transcribed into mRNA and translated into protein.

[0096] A cell has been “stably transfected” with a nucleic acidconstruct when the nucleic acid construct is capable of being inheritedby daughter cells.

[0097] As used herein, a “reporter gene construct” is a nucleic acidthat includes a “reporter gene” operatively linked to at least onetranscriptional regulatory sequence. Transcription of the reporter geneis controlled by these sequences to which they are linked. The activityof at least one or more of these control sequences can be directly orindirectly regulated by the target receptor protein. Exemplarytranscriptional control sequences are promoter sequences. A reportergene is meant to include a promoter-reporter gene construct that isheterologously expressed in a cell.

[0098] As used herein, “transformed cells” refers to cells that havespontaneously converted to a state of unrestrained growth, i.e., theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control. For purposes of this invention, the terms“transformed phenotype of malignant mammalian cells” and “transformedphenotype ” are intended to encompass, but not be limited to, any of thefollowing phenotypic traits associated with cellular transformation ofmammalian cells: immortalization, morphological or growthtransformation, and tumorigenicity, as detected by prolonged growth incell culture, growth in semi-solid media, or tumorigenic growth inimmuno-incompetent or syngeneic animals.

[0099] As used herein, “proliferating” and “proliferation” refer tocells undergoing mitosis.

[0100] As used herein, “immortalized cells” refers to cells that havebeen altered via chemical, genetic, and/or recombinant means such thatthe cells have the ability to grow through an indefinite number ofdivisions in culture.

[0101] The “growth state” of a cell refers to the rate of proliferationof the cell and the state of differentiation of the cell.

III. Exemplary Embodiments of Isolation Method

[0102] One aspect of the invention provides a method for potentiatingRNAi by induction or ectopic activation of an RNAi enzyme in a cell (invivo or in vitro) or cell-free mixtures. In preferred embodiments, theRNAi activity is activated or added to a mammalian cell, e.g., a humancell, which cell may be provided in vitro or as part of a wholeorganism. In other embodiments, the subject method is carried out usingeukaryotic cells generally (except for oocytes) in culture. Forinstance, the Dicer enzyme may be activated by virtue of beingrecombinantly expressed or it may be activated by use of an agent which(i) induces expression of the endogenous gene, (ii) stabilizes theprotein from degradation, and/or (iii) allosterically modies the enzymeto increase its activity (by altering its Kcat, Km or both).

[0103] A. Dicer and Argonaut Activities

[0104] In certain embodiment, at least one of the activated RNAi enzymesis Dicer, or a homolog thereof. In certain preferred embodiments, thepresent method provides for ectopic activation of Dicer. As used herein,the term “Dicer” refers to a protein which (a) mediates an RNAi responseand (b) has an amino acid sequence at least 50 percent identical, andmore preferablty at least 75, 85, 90 or 95 percent identical to SEQ IDNo. 2 or 4, and/or which can be encoded by a nucleic acid whichhybridizes under wash conditions of 2×SSC at 22° C., and more preferably0.2×SSC at 65° C., to a nucleotide represented by SEQ ID No. 1 or 3.Accordingly, the method may comprise introducing a dsRNA contruct into acell in which Dicer has been recombinantly expressed or otherwiseectopically activated.

[0105] In certain embodiment, at least one of the activated RNAi enzymesis Argonaut, or a homolog thereof. In certain preferred embodiments, thepresent method provides for ectopic activation of Argonaut. As usedherein, the term “Argonaut” refers to a protein which (a) mediates anRNAi response and (b) has an amino acid sequence at least 50 percentidentical, and more preferablty at least 75, 85, 90 or 95 percentidentical to the amino acid sequence shown in FIG. 24. Accordingly, themethod may comprise introducing a dsRNA contruct into a cell in whichArgonaut has been recombinantly expressed or otherwise ectopicallyactivated.

[0106] This invention also provides expression vectors containing anucleic acid encoding a Dicer or Argonaut polypeptides, operably linkedto at least one transcriptional regulatory sequence. Operably linked isintended to mean that the nucleotide sequence is linked to a regulatorysequence in a manner which allows expression of the nucleotide sequence.Regulatory sequences are art-recognized and are selected to directexpression of the subject Dicer or Argonaut proteins. Accordingly, theterm transcriptional regulatory sequence includes promoters, enhancersand other expression control elements. Such regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). For instance, any of awide variety of expression control sequences, sequences that control theexpression of a DNA sequence when operatively linked to it, may be usedin these vectors to express DNA sequences encoding Dicer or Argonautpolypeptides of this invention. Such useful expression controlsequences, include, for example, a viral LTR, such as the LTR of theMoloney murine leukemia virus, the early and late promoters of SV40,adenovirus or cytomegalovirus immediate early promoter, the lac system,the trp system, the TAC or TRC system, T7 promoter whose expression isdirected by T7 RNA polymerase, the major operator and promoter regionsof phage λ, the control regions for fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast α-matingfactors, the polyhedron promoter of the baculovirus system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed.

[0107] Moreover, the vector's copy number, the ability to control thatcopy number and the expression of any other proteins encoded by thevector, such as antibiotic markers, should also be considered.

[0108] The recombinant Dicer or Argonaut genes can be produced byligating nucleic acid encoding a Dicer or Argonaut polypeptide into avector suitable for expression in either prokaryotic cells, eukaryoticcells, or both. Expression vectors for production of recombinant formsof the subject Dicer or Argonaut polypeptides include plasmids and othervectors. For instance, suitable vectors for the expression of a Dicer orArgonaut polypeptide include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

[0109] A number of vectors exist for the expression of recombinantproteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, andYRP17 are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein). These vectorscan replicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, a Dicer or Argonaut polypeptide isproduced recombinantly utilizing an expression vector generated bysub-cloning the coding sequence of a Dicer or Argonaut gene.

[0110] The preferred mammalian expression vectors contain bothprokaryotic sequences, to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and17.

[0111] In yet another embodiment, the subject invention provides a “geneactivation” construct which, by homologous recombination with a genomicDNA, alters the transcriptional regulatory sequences of an endogenousDicer or Argonaut gene. For instance, the gene activation construct canreplace the endogenous promoter of a Dicer or Argonaut gene with aheterologous promoter, e.g., one which causes constitutive expression ofthe Dicer or Argonaut gene or which causes inducible expression of thegene under conditions different from the normal expression pattern ofDicer or Argonaut. A variety of different formats for the geneactivation constructs are available. See, for example, the TranskaryoticTherapies, Inc PCT publications WO93/09222, WO95/31560, WO96/29411,WO95/31560 and WO94/12650.

[0112] In preferred embodiments, the nucleotide sequence used as thegene activation construct can be comprised of (1) DNA from some portionof the endogenous Dicer or Argonaut gene (exon sequence, intronsequence, promoter sequences, etc.) which direct recombination and (2)heterologous transcriptional regulatory sequence(s) which is to beoperably linked to the coding sequence for the genomic Dicer or Argonautgene upon recombination of the gene activation construct. For use ingenerating cultures of Dicer or Argonaut producing cells, the constructmay further include a reporter gene to detect the presence of theknockout construct in the cell.

[0113] The gene activation construct is inserted into a cell, andintegrates with the genomic DNA of the cell in such a position so as toprovide the heterologous regulatory sequences in operative associationwith the native Dicer or Argonaut gene. Such insertion occurs byhomologous recombination, i.e., recombination regions of the activationconstruct that are homologous to the endogenous Dicer or Argonaut genesequence hybridize to the genomic DNA and recombine with the genomicsequences so that the construct is incorporated into the correspondingposition of the genomic DNA.

[0114] The terms “recombination region” or “targeting sequence” refer toa segment (i.e., a portion) of a gene activation construct having asequence that is substantially identical to or substantiallycomplementary to a genomic gene sequence, e.g., including 5′ flankingsequences of the genomic gene, and can facilitate homologousrecombination between the genomic sequence and the targeting transgeneconstruct.

[0115] As used herein, the term “replacement region” refers to a portionof a activation construct which becomes integrated into an endogenouschromosomal location following homologous recombination between arecombination region and a genomic sequence.

[0116] The heterologous regulatory sequences, e.g., which are providedin the replacement region, can include one or more of a varietyelements, including: promoters (such as constitutive or induciblepromoters), enhancers, negative regulatory elements, locus controlregions, transcription factor binding sites, or combinations thereof.

[0117] Promoters/enhancers which may be used to control expression ofthe targeted gene in vivo include, but are not limited to, thecytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989, J.Exp. Med., 169:13), the human β-actin promoter (Gunning et al. (1987)PNAS 84:4831-4835), the glucocorticoid-inducible promoter present in themouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig etal. (1984) Mol. Cell Biol. 4:1354-1362), the long terminal repeatsequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al.(1985) RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.), the SV40 early or late region promoter (Bernoist et al.(1981) Nature 290:304-310; Templeton et al. (1984) Mol. Cell Biol.,4:817; and Sprague et al. (1983) J. Virol., 45:773), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (RSV)(Yamamoto et al., 1980, Cell, 22:787-797), the herpes simplex virus(HSV) thymidine kinase promoter/enhancer (Wagner et al. (1981) PNAS82:3567-71), and the herpes simplex virus LAT promoter (Wolfe et al.(1992) Nature Genetics, 1:379-384).

[0118] In still other embodiments, the replacement region merely deletesa negative transcriptional control element of the native gene, e.g., toactivate expression, or ablates a positive control element, e.g., toinhibit expression of the targeted gene.

[0119] B. Cell/Organism

[0120] The cell with the target gene may be derived from or contained inany organism (e.g., plant, animal, protozoan, virus, bacterium, orfungus). The dsRNA construct may be synthesized either in vivo or invitro. Endogenous RNA polymerase of the cell may mediate transcriptionin vivo, or cloned RNA polymerase can be used for transcription in vivoor in vitro. For generating double stranded transcripts from a transgenein vivo, a regulatory region may be used to transcribe the RNA strand(or strands).

[0121] Furthermore, genetic manipulation becomes possible in organismsthat are not classical genetic models. Breeding and screening programsmay be accelerated by the ability to rapidly assay the consequences of aspecific, targeted gene disruption. Gene disruptions may be used todiscover the function of the target gene, to produce disease models inwhich the target gene are involved in causing or preventing apathological condition, and to produce organisms with improved economicproperties.

[0122] The cell with the target gene may be derived from or contained inany organism. The organism may a plant, animal, protozoan, bacterium,virus, or fungus. The plant may be a monocot, dicot or gymnosperm; theanimal may be a vertebrate or invertebrate. Preferred microbes are thoseused in agriculture or by industry, and those that are pathogenic forplants or animals. Fungi include organisms in both the mold and yeastmorphologies.

[0123] Plants include arabidopsis; field crops (e.g., alfalfa, barley,bean, com, cotton, flax, pea, rape, rice, rye, safflower, sorghum,soybean, sunflower, tobacco, and wheat); vegetable crops (e.g.,asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery,cucumber, eggplant, lettuce, onion, pepper, potato, pumpkin, radish,spinach, squash, taro, tomato, and zucchini); fruit and nut crops (e.g.,almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry,coconut, cranberry, date, faJoa, filbert, grape, grapefruit, guava,kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passionfruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry,strawberry, tangerine, walnut, and watermelon); and ornamentals (e.g.,alder, ash, aspen, azalea, birch, boxwood, camellia, carnation,chrysanthemum, elm, fir, ivy, jasmine, juniper, oak, palm, poplar, pine,redwood, rhododendron, rose, and rubber).

[0124] Examples of vertebrate animals include fish, mammal, cattle,goat, pig, sheep, rodent, hamster, mouse, rat, primate, and human.

[0125] Invertebrate animals include nematodes, other worms, drosophila,and other insects. Representative generae of nematodes include thosethat infect animals (e.g., Ancylostoma, Ascaridia, Ascaris, Bunostomum,Caenorhabditis, Capillaria, Chabertia, Cooperia, Dictyocaulus,Haernonchus, Heterakis, Nematodirus, Oesophagostomum, Ostertagia,Oxyuris, Parascaris, Strongylus, Toxascaris, Trichuris,Trichostrongylus, Tflichonema, Toxocara, Uncinaria) and those thatinfect plants (e.g., B ursaphalenchus, Criconerriella, Diiylenchus,Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus,Melodoigyne, Nacobbus, Paratylenchus, Pratylenchus, Radopholus,Rotelynchus, Tylenchus, and Xiphinerna). Representative orders ofinsects include Coleoptera, Diptera, Lepidoptera, and Homoptera.

[0126] The cell having the target gene may be from the germ line orsomatic, totipotent or pluripotent, dividing or non-dividing, parenchymaor epithelium, immortalized or transformed, or the like. The cell may bea stem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

[0127] C. Targeted Genes

[0128] The target gene may be a gene derived from the cell, anendogenous gene, a transgene, or a gene of a pathogen which is presentin the cell after infection thereof. Depending on the particular targetgene and the dose of double stranded RNA material delivered, theprocedure may provide partial or complete loss of function for thetarget gene. Lower doses of injected material and longer times afteradministration of dsRNA may result in inhibition in a smaller fractionof cells. Quantitation of gene expression in a cell may show similaramounts of inhibition at the level of accumulation of target mRNA ortranslation of target protein.

[0129] “Inhibition of gene expression” refers to the absence (orobservable decrease) in the level of protein and/or mRNA product from atarget gene. “Specificity” refers to the ability to inhibit the targetgene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioImmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS). For RNA-mediated inhibition in a cell line orwhole organism, gene expression is conveniently assayed by use of areporter or drug resistance gene whose protein product is easilyassayed. Such reporter genes include acetohydroxyacid synthase (AHAS),alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase(GUS), chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopalinesynthase (NOS), octopine synthase (OCS), and derivatives thereofmultiple selectable markers are available that confer resistance toampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, andtetracyclin.

[0130] Depending on the assay, quantitation of the amount of geneexpression allows one to determine a degree of inhibition which isgreater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell nottreated according to the present invention. Lower doses of injectedmaterial and longer times after administration of dsRNA may result ininhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%,75%,90%, or 95% of targeted cells). Quantitation of gene expression in acell may show similar amounts of inhibition at the level of accumulationof target mRNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell: mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

[0131] As disclosed herein, the present invention may is not limited toany type of target gene or nucleotide sequence. But the followingclasses of possible target genes are listed for illustrative purposes:developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors,Writ family members, Pax family members, Winged helix family members,Hox family members, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2,CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETS1, ETV6, FGR, FOS, FYN, HCR,HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor genes (e.g.,APC, BRCA 1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI); andenzymes (e.g., ACC synthases and oxidases, ACP desaturases andhydroxylases, ADP-glucose pyrophorylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextrinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hemicellulases, integrases, inulinases, invertases, isomerases, kinases,lactases, lipases, lipoxygenases, lysozymes, nopaline synthases,octopine synthases, pectinesterases, peroxidases, phosphatases,phospholipases, phosphorylases, phytases, plant growth regulatorsynthases, polygalacturonases, proteinases and peptidases, pullanases,recombinases, reverse transcriptases, RUBISCOs, topoisomerases, andxylanases).

[0132] D. dsRNA constructs

[0133] The dsRNA construct may comprise one or more strands ofpolymerized ribonucleotide. It may include modifications to either thephosphate-sugar backbone or the nucleoside. For example, thephosphodiester linkages of natural RNA may be modified to include atleast one of a nitrogen or sulfur heteroatom. Modifications in RNAstructure may be tailored to allow specific genetic inhibition whileavoiding a general panic response in some organisms which is generatedby dsRNA. Likewise, bases may be modified to block the activity ofadenosine deaminase. The dsRNA construct may be produced enzymaticallyor by partial/total organic synthesis, any modified ribonucleotide canbe introduced by in vitro enzymatic or organic synthesis.

[0134] The dsRNA construct may be directly introduced into the cell(i.e., intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing an organism in a solutioncontaining RNA. Methods for oral introduction include direct mixing ofRNA with food of the organism, as well as engineered approaches in whicha species that is used as food is engineered to express an RNA, then fedto the organism to be affected. Physical methods of introducing nucleic,acids include injection directly into the cell or extracellularinjection into the organism of an RNA solution.

[0135] The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of double-stranded material may yield more effectiveinhibition; lower doses may also be useful for specific applications.Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition.

[0136] dsRNA constructs containing a nucleotide sequences identical to aportion of the target gene are preferred for inhibition. RNA sequenceswith insertions, deletions, and single point mutations relative to thetarget sequence have also been found to be effective for inhibition.Thus, sequence identity may optimized by sequence comparison andalignment algorithms known in the art (see Gribskov and Devereux,Sequence Analysis Primer, Stockton Press, 1991, and references citedtherein) and calculating the percent difference between the nucleotidesequences by, for example, the Smith-Waterman algorithm as implementedin the BESTFIT software program using default parameters (e.g.,University of Wisconsin Genetic Computing Group). Greater than 90%sequence identity, or even 100% sequence identity, between theinhibitory RNA and the portion of the target gene is preferred.Alternatively, the duplex region of the RNA may be defined functionallyas a nucleotide sequence that is capable of hybridizing with a portionof the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed bywashing). In certain preferred embodiments, the length of the dsRNA isat least 20, 21 or 22 nucleotides in length, e.g., corresponding in sizeto RNA products produced by Dicer-dependent cleavage. In certainembodiments, the dsRNA construct is at least 25, 50, 100, 200, 300 or400 bases. In certain embodiments, the dsRNA construct is 400-800 basesin length.

[0137] 100% sequence identity between the RNA and the target gene is notrequired to practice the present invention. Thus the invention has theadvantage of being able to tolerate sequence variations that might beexpected due to genetic mutation, strain polymorphism, or evolutionarydivergence.

[0138] The dsRNA construct may be synthesized either in vivo or invitro. Endogenous RNA polymerase of the cell may mediate transcriptionin vivo, or cloned RNA polymerase can be used for transcription in vivoor in vitro. For transcription from a transgene in vivo or an expressionconstruct, a regulatory region (e.g., promoter, enhancer, silencer,splice donor and acceptor, polyadenylation) may be used to transcribethe dsRNA strand (or strands). Inhibition may be targeted by specifictranscription in an organ, tissue, or cell type; stimulation of anenvironmental condition (e.g., infection, stress, temperature, chemicalinducers); and/or engineering transcription at a developmental stage orage. The RNA strands may or may not be polyadenylated; the RNA strandsmay or may not be capable of being translated into a polypeptide by acell's translational apparatus. The dsRNA construct may be chemically orenzymatically synthesized by manual or automated reactions. The dsRNAconstruct may be synthesized by a cellular RNA polymerase or abacteriophage RNA polymerase (e.g., T3, T7, SP6). The use and productionof an expression construct are known in the art 32,33,34 (see also WO97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and5,804,693; and the references cited therein). If synthesized chemicallyor by in vitro enzymatic synthesis, the RNA may be purified prior tointroduction into the cell. For example, RNA can be punified from amixture by extraction with a solvent or resin, precipitation,electrophoresis, chromatography or a combination thereof. Alternatively,the dsRNA construct may be used with no or a minimum of purification toavoid losses due to sample processing. The dsRNA construct may be driedfor storage or dissolved in an aqueous solution. The solution maycontain buffers or salts to promote annealing, and/or stabilization ofthe duplex strands.

[0139] Physical methods of introducing nucleic acids include injectionof a solution containing the dsRNA construct, bombardment by particlescovered by the dsRNA construct, soaking the cell or organism in asolution of the RNA, or electroporation of cell membranes in thepresence of the dsRNA construct. A viral construct packaged into a viralparticle would accomplish both efficient introduction of an expressionconstruct into the cell and transcription of dsRNA construct encoded bythe expression construct. Other methods known in the art for introducingnucleic acids to cells may be used, such as lipid-mediated carriertransport, chemicalmediated transport, such as calcium phosphate, andthe like. Thus the dsRNA construct may be introduced along withcomponents that perform one or more of the following activities: enhanceRNA uptake by the cell, promote annealing of the duplex strands,stabilize the annealed strands, or other-wise increase inhibition of thetarget gene.

[0140] E. Illustrative Uses

[0141] One utility of the present invention is as a method ofidentifying gene function in an organism, especially higher eukaryotescomprising the use of double-stranded RNA to inhibit the activity of atarget gene of previously unknown function. Instead of the timeconsuming and laborious isolation of mutants by traditional geneticscreening, functional genomics would envision determining the functionof uncharacterized genes by employing the invention to reduce the amountand/or alter the timing of target gene activity. The invention could beused in determining potential targets for pharmaceutics, understandingnormal and pathological events associated with development, determiningsignaling pathways responsible for postnatal development/aging, and thelike. The increasing speed of acquiring nucleotide sequence informationfrom genomic and expressed gene sources, including total sequences formammalian genomes, can be coupled with the invention to determine genefunction in a cell or in a whole organism. The preference of differentorganisms to use particular codons, searching sequence databases forrelated gene products, correlating the linkage map of genetic traitswith the physical map from which the nucleotide sequences are derived,and artificial intelligence methods may be used to define putative openreading frames from the nucleotide sequences acquired in such sequencingprojects.

[0142] A simple assay would be to inhibit gene expression according tothe partial sequence available from an expressed sequence tag (EST).Functional alterations in growth, development, metabolism, diseaseresistance, or other biological processes would be indicative of thenormal role of the EST's gene product.

[0143] The ease with which the dsRNA construct can be introduced into anintact cell/organism containing the target gene allows the presentinvention to be used in high throughput screening (HTS). For example,duplex RNA can be produced by an amplification reaction using primersflanking the inserts of any gene library derived from the target cell ororganism. Inserts may be derived from genomic DNA or mRNA (e.g., cDNAand cRNA). Individual clones from the library can be replicated and thenisolated in separate reactions, but preferably the library is maintainedin individual reaction vessels (e.g., a 96 well microtiter plate) tominimize the number of steps required to practice the invention and toallow automation of the process.

[0144] In an exemplary embodiment, the subject invention provides anarrayed library of RNAi constructs. The array may in the form ofsolutions, such as multi-well plates, or may be “printed” on solidsubstrates upon which cells can be grown. To illustrate, solutionscontaining duplex RNAs that are capable of inhibiting the differentexpressed genes can be placed into individual wells positioned on amicrotiter plate as an ordered array, and intact cells/organisms in eachwell can be assayed for any changes or modifications in behavior ordevelopment due to inhibition of target gene activity.

[0145] In one embodiment, the subject method uses an arrayed library ofRNAi constructs to screen for combinations of RNAi that is lethal tohost cells. Synthetic lethality is a bedrock principle of experimentalgenetics. A synthetic lethality describes the properties of twomutations which, individually, are tolerated by the organism but which,in combination, are lethal. The subject arrays can be used to identifyloss-of-function mutations that are lethal in combination withalterations in other genes, such as activated oncogenes orloss-of-function mutations to tumor suppressors. To achieve this, onecan create “phenotype arrays” using cultured cells. Expression of eachof a set of genes, such as the host cell's genome, can be individuallysystematically disrupted using RNA interference. Combination withalterations in oncogene and tumor suppressor pathways can be used toidentify synthetic lethal interactions that may identify noveltherapeutic targets.

[0146] In certain embodiments, the RNAi constructs can be fed directlyto, injected into, the cell/organism containing the target gene.Alternatively, the duplex RNA can be produced by in vivo or in vitrotranscription from an expression construct used to produce the library.The construct can be replicated as individual clones of the library andtranscribed to produce the RNA; each clone can then be fed to, orinjected into, the cell/organism containing the target gene. Thefunction of the target gene can be assayed from the effects it has onthe cell/organism when gene activity is inhibited. This screening couldbe amenable to small subjects that can be processed in large number, forexample, tissue culture cells derived from mammals, especially primates,and most preferably humans.

[0147] If a characteristic of an organism is determined to begenetically linked to a polymorphism through RFLP or QTL analysis, thepresent invention can be used to gain insight regarding whether thatgenetic polymorphism might be directly responsible for thecharacteristic. For example, a fragment defining the geneticpolymorphism or sequences in the vicinity of such a genetic polymorphismcan be amplified to produce an RNA, the duplex RNA can be introduced tothe organism or cell, and whether an alteration in the charactenstic iscorrelated with inhibition can be determined. Of course, there may betrivial explanations for negative results with this type of assay, forexample: inhibition of the target gene causes lethality, inhibition ofthe target gene may not result in any observable alteration, thefragment contains nucleotide sequences that are not capable ofinhibiting the target gene, or the target gene's activity is redundant.

[0148] The present invention may be useful in allowing the inhibition ofessential genes. Such genes may be required for cell or organismviability at only particular stages of development or cellularcompartments. The functional equivalent of conditional mutations may beproduced by inhibiting activity of the target gene when or where it isnot required for viability. The invention allows addition of RNA atspecific times of development and locations in the organism withoutintroducing permanent mutations into the target genome.

[0149] If alternative splicing produced a family of transcripts thatwere distinguished by usage of characteristic exons, the presentinvention can target inhibition through the appropriate exons tospecifically inhibit or to distinguish among the functions of familymembers. For example, a hormone that contained an alternatively splicedtransmembrane domain may be expressed in both membrane bound andsecreted forms. Instead of isolating a nonsense mutation that terminatestranslation before the transmembrane domain, the functional consequencesof having only secreted hormone can be determined according to theinvention by targeting the exon containing the transmembrane domain andthereby inhibiting expression of membrane-bound hormone.

[0150] The present invention may be used alone or as a component of akit having at least one of the reagents necessary to carry out the invitro or in vivo introduction of RNA to test samples or subjects.Preferred components are the dsRNA and a vehicle that promotesintroduction of the dsRNA. Such a kit may also include instructions toallow a user of the kit to practice the invention.

[0151] Alternatively, an organism may be engineered to produce dsRNAwhich produces commercially or medically beneficial results, forexample, resistance to a pathogen or its pathogenic effects, improvedgrowth, or novel developmental patterns.

IV. Exemplification

[0152] The invention, now being generally described, will be morereadily understood by reference to the following examples, which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention and are not intended to limit theinvention.

Example 1 An RNA-directed Nuclease Mediates RNAi Gene Silencing

[0153] In a diverse group of organisms that includes Caenorhabditiselegans, Drosophila, planaria, hydra, trypanosomes, fungi and plants,the introduction of double-stranded RNAs inhibits gene expression in asequence-specific manner¹⁻⁷. These responses, called RNA interference orpost-transcriptional gene silencing, may provide anti-viral defence,modulate transposition or regulate gene expression^(1, 6, 8-10). We havetaken a biochemical approach towards elucidating the mechanismsunderlying this genetic phenomenon. Here we show that ‘loss-of-function’phenotypes can be created in cultured Drosophila cells by transfectionwith specific double-stranded RNAs. This coincides with a markedreduction in the level of cognate cellular messenger RNAs. Extracts oftransfected cells contain a nuclease activity that specifically degradesexogenous transcripts homologous to transfected double-stranded RNA.This enzyme contains an essential RNA component. After partialpurification, the sequence-specific nuclease co-fractionates with adiscrete, ˜25-nucleotide RNA species which may confer specificity to theenzyme through homology to the substrate mRNAs.

[0154] Although double-stranded RNAs (dsRNAs) can provoke gene silencingin numerous biological contexts including Drosophila^(11, 12), themechanisms underlying this phenomenon have remained mostly unknown. Wetherefore wanted to establish a biochemically tractable model in whichsuch mechanisms could be investigated.

[0155] Transient transfection of cultured, Drosophila S2 cells with alacZ expression vector resulted in β-galactosidase activity that waseasily detectable by an in situ assay (FIG. 1a). This activity wasgreatly reduced by co-transfection with a dsRNA corresponding to thefirst 300 nucleotides of the lacZ sequence, whereas co-transfection witha control dsRNA (CD8) (FIG. 1a) or with single-stranded RNAs of eithersense or antisense orientation (data not shown) had little or no effect.This indicated that dsRNAs could interfere, in a sequence-specificfashion, with gene expression in cultured cells.

[0156] To determine whether RNA interference (RNAi) could be used totarget endogenous genes, we transfected S2 cells with a dsRNAcorresponding to the first 540 nucleotides of Drosophila cyclin E, agene that is essential for progression into S phase of the cell cycle.During log-phase growth, untreated S2 cells reside primarily in G2/M(FIG. 1b). Transfection with lacZ dsRNA had no effect on cell-cycledistribution, but transfection with the cyclin E dsRNA caused a G1-phasecell-cycle arrest (FIG. 1b). The ability of cyclin E dsRNA to provokethis response was length-dependent. Double-stranded RNAs of 540 and 400nucleotides were quite effective, whereas dsRNAs of 200 and 300nucleotides were less potent. Double-stranded cyclin E RNAs of 50 or 100nucleotides were inert in our assay, and transfection with asingle-stranded, antisense cyclin E RNA had virtually no effect.

[0157] One hallmark of RNAi is a reduction in the level of mRNAs thatare homologous to the dsRNA. Cells transfected with the cyclin E dsRNA(bulk population) showed diminished endogenous cyclin E mRNA as comparedwith control cells (FIG. 1c). Similarly, transfection of cells withdsRNAs homologous to fizzy, a component of the anaphase-promotingcomplex (APC) or cyclin A, a cyclin that acts in S, G2 and M, alsocaused reduction of their cognate mRNAs (FIG. 1c). The modest reductionin fizzy mRNA levels in cells transfected with cyclin A dsRNA probablyresulted from arrest at a point in the division cycle at which fizzytranscription is low^(14, 15). These results indicate that RNAi may be agenerally applicable method for probing gene function in culturedDrosophila cells.

[0158] The decrease in mRNA levels observed upon transfection ofspecific dsRNAs into Drosophila cells could be explained by effects attranscriptional or post-transcriptional levels. Data from other systemshave indicated that some elements of the dsRNA response may affect mRNAdirectly (reviewed in refs 1 and 6). We therefore sought to develop acell-free assay that reflected, at least in part, RNAi.

[0159] S2 cells were transfected with dsRNAs corresponding to eithercyclin E or lacZ. Cellular extracts were incubated with synthetic mRNAsof lacZ or cyclin E. Extracts prepared from cells transfected with the540-nucleotide cyclin E dsRNA efficiently degraded the cyclin Etranscript; however, the lacZ transcript was stable in these lysates(FIG. 2a). Conversely, lysates from cells transfected with the lacZdsRNA degraded the lacZ transcript but left the cyclin E mRNA intact.These results indicate that RNAi ablates target mRNAs through thegeneration of a sequence-specific nuclease activity. We have termed thisenzyme RISC (RNA-induced silencing complex). Although we occasionallyobserved possible intermediates in the degradation process (see FIG. 2),the absence of stable cleavage end-products indicates an exonuclease(perhaps coupled to an endonuclease). However, it is possible that theRNAi nuclease makes an initial endonucleolytic cut and that non-specificexonucleases in the extract complete the degradation process¹⁶. Inaddition, our ability to create an extract that targets lacZ in vitroindicates that the presence of an endogenous gene is not required forthe RNAi response.

[0160] To examine the substrate requirements for the dsRNA-induced,sequence-specific nuclease activity, we incubated a variety ofcyclin-E-derived transcripts with an extract derived from cells that hadbeen transfected with the 540-nucleotide cyclin E dsRNA (FIG. 2b, c).Just as a length requirement was observed for the transfected dsRNA, theRNAi nuclease activity showed a dependence on the size of the RNAsubstrate. Both a 600-nucleotide transcript that extends slightly beyondthe targeted region (FIG. 2b) and an ˜1-kilobase (kb) transcript thatcontains the entire coding sequence (data not shown) were completelydestroyed by the extract. Surprisingly, shorter substrates were notdegraded as efficiently. Reduced activity was observed against either a300- or a 220-nucleotide transcript, and a 100-nucleotide transcript wasresistant to nuclease in our assay. This was not due solely to positioneffects because ˜100-nucleotide transcripts derived from other portionsof the transfected dsRNA behaved similarly (data not shown). Asexpected, the nuclease activity (or activities) present in the extractcould also recognize the antisense strand of the cyclin E mRNA. Again,substrates that contained a substantial portion of the targeted regionwere degraded efficiently whereas those that contained a shorter stretchof homologous sequence (˜130 nucleotides) were recognized inefficiently(FIG. 2c, as600). For both the sense and antisense strands, transcriptsthat had no homology with the transfected dsRNA ( FIG. 2b, Eout; FIG.2c, as300) were not degraded. Although we cannot exclude the possibilitythat nuclease specificity could have migrated beyond the targetedregion, the resistance of transcripts that do not contain homology tothe dsRNA is consistent with data from C. elegans. Double-stranded RNAshomologous to an upstream cistron have little or no effect on a linkeddownstream cistron, despite the fact that unprocessed, polycistronicmRNAs can be readily detected^(17, 18). Furthermore, the nuclease wasinactive against a dsRNA identical to that used to provoke the RNAiresponse in vivo (FIG. 2b). In the in vitro system, neither a 5′ cap nora poly(A) tail was required, as such transcripts were degraded asefficiently as uncapped and non-polyadenylated RNAs.

[0161] Gene silencing provoked by dsRNA is sequence specific. Aplausible mechanism for determining specificity would be incorporationof nucleic-acid guide sequences into the complexes that accomplishsilencing¹⁹. In accord with this idea, pre-treatment of extracts with aCa²⁺-dependent nuclease (micrococcal nuclease) abolished the ability ofthese extracts to degrade cognate mRNAs (FIG. 3). Activity could not berescued by addition of non-specific RNAs such as yeast transfer RNA.Although micrococcal nuclease can degrade both DNA and RNA, treatment ofthe extract with DNAse I had no effect (FIG. 3). Sequence-specificnuclease activity, however, did require protein (data not shown).Together, our results support the possibility that the RNAi nuclease isa ribonucleoprotein, requiring both RNA and protein components.Biochemical fractionation (see below) is consistent with thesecomponents being associated in extract rather than being assembled onthe target mRNA after its addition.

[0162] In plants, the phenomenon of co-suppression has been associatedwith the existence of small (˜25-nucleotide) RNAs that correspond to thegene that is being silenced¹⁹. To address the possibility that a similarRNA might exist in Drosophila and guide the sequence-specific nucleasein the choice of substrate, we partially purified our activity throughseveral fractionation steps. Crude extracts contained bothsequence-specific nuclease activity and abundant, heterogeneous RNAshomologous to the transfected dsRNA (FIGS. 2 and 4a). The RNAi nucleasefractionated with ribosomes in a high-speed centrifugation step.Activity could be extracted by treatment with high salt, and ribosomescould be removed by an additional centrifugation step. Chromatography ofsoluble nuclease over an anion-exchange column resulted in a discretepeak of activity (FIG. 4b, cyclin E). This retained specificity as itwas inactive against a heterologous mRNA (FIG. 4b, lacZ). Activefractions also contained an RNA species of 25 nucleotides that ishomologous to the cyclin E target (FIG. 4b, northern). The band observedon northern blots may represent a family of discrete RNAs because itcould be detected with probes specific for both the sense and antisensecyclin E sequences and with probes derived from distinct segments of thedsRNA (data not shown). At present, we cannot determine whether the25-nucleotide RNA is present in the nuclease complex in adouble-stranded or single-stranded form.

[0163] RNA interference allows an adaptive defence against bothexogenous and endogenous dsRNAs, providing something akin to a dsRNAimmune response. Our data, and that of others¹⁹, is consistent with amodel in which dsRNAs present in a cell are converted, either throughprocessing or replication, into small specificity determinants ofdiscrete size in a manner analogous to antigen processing. Our resultssuggest that the post-transcriptional component of dsRNA-dependent genesilencing is accomplished by a sequence-specific nuclease thatincorporates these small RNAs as guides that target specific messagesbased upon sequence recognition. The identical size of putativespecificity determinants in plants¹⁹ and animals predicts a conservationof both the mechanisms and the components of dsRNA-induced,post-transcriptional gene silencing in diverse organisms. In plants,dsRNAs provoke not only post-transcriptional gene silencing but alsochromatin remodelling and transcriptional repression^(20, 21). It is nowcritical to determine whether conservation of gene-silencing mechanismsalso exists at the transcriptional level and whether chromatinremodelling can be directed in a sequence-specific fashion by these samedsRNA-derived guide sequences.

[0164] Methods

[0165] Cell Culture and RNA Methods

[0166] S2 (ref. 22) cells were cultured at 27° C. in 90% Schneider'sinsect media (Sigma), 10% heat inactivated fetal bovine serum (FBS).Cells were transfected with dsRNA and plasmid DNA by calcium phosphateco-precipitation²³. Identical results were observed when cells weretransfected using lipid reagents (for example, Superfect, Qiagen). ForFACS analysis, cells were additionally transfected with a vector thatdirects expression of a green fluorescent protein (GFP)-US9 fusionprotein¹³. These cells were fixed in 90% ice-cold ethanol and stainedwith propidium iodide at 25 μg ml⁻¹. FACS was performed on an Elite flowcytometer (Coulter). For northern blotting, equal loading was ensured byover-probing blots with a control complementary DNA (RP49). For theproduction of dsRNA, transcription templates were generated bypolymerase chain reaction such that they contained T7 promoter sequenceson each end of the template. RNA was prepared using the RiboMax kit(Promega). Confirmation that RNAs were double stranded came from theircomplete sensitivity to RNAse III (a gift from A. Nicholson). TargetmRNA transcripts were synthesized using the Riboprobe kit (Promega) andwere gel purified before use.

[0167] Extract Preparation

[0168] Log-phase S2 cells were plated on 15-cm tissue culture dishes andtransfected with 30 μg dsRNA and 30 μg carrier plasmid DNA. Seventy-twohours after transfection, cells were harvested in PBS containing 5 mMEGTA washed twice in PBS and once in hypotonic buffer (10 mM HEPES pH7.3, 6 mM β-mercaptoethanol). Cells were suspended in 0.7 packed-cellvolumes of hypotonic buffer containing Complete protease inhibitors(Boehringer) and 0.5 units ml⁻¹ of RNasin (Promega). Cells weredisrupted in a dounce homogenizer with a type B pestle, and lysates werecentrifuged at 30,000 g for 20 min. Supernatants were used in an invitro assay containing 20 mM HEPES pH 7.3, 110 mM KOAc, 1 mM Mg(OAc)₂, 3mM EGTA, 2 mM CaCl₂, 1 mM DTT. Typically, 5 μl extract was used in a 10μl assay that contained also 10,000 c.p.m. synthetic mRNA substrate.

[0169] Extract Fractionation

[0170] Extracts were centrifuged at 200,000 g for 3 h and the resultingpellet (containing ribosomes) was extracted in hypotonic buffercontaining also 1 mM MgCl₂ and 300 mM KOAc. The extracted material wasspun at 100,000 g for 1 h and the resulting supernatant was fractionatedon Source 15Q column (Pharmacia) using a KCl gradient in buffer A (20 mMHEPES pH 7.0, 1 mM dithiothreitol, 1 mM MgCl₂). Fractions were assayedfor nuclease activity as described above. For northern blotting,fractions were proteinase K/SDS treated, phenol extracted, and resolvedon 15% acrylamide 8M urea gels. RNA was electroblotted onto Hybond N+and probed with strand-specific riboprobes derived from cyclin E mRNA.Hybridization was carried out in 500 mM NaPO₄ pH 7.0, 15% formamide, 7%SDS, 1% BSA. Blots were washed in 1 SSC at 37-45° C.

References Cited in Example 1

[0171] 1. Sharp, P. A. RNAi and double-strand RNA. Genes Dev. 13,139-141 (1999).

[0172] 2. Sanchez-Alvarado, A. & Newmark, P. A. Double-stranded RNAspecifically disrupts gene expression during planarian regeneration.Proc. Natl Acad. Sci. USA 96, 5049-5054 (1999).

[0173] 3. Lohmann, J. U., Endl, I. & Bosch, T. C. Silencing ofdevelopmental genes in Hydra. Dev. Biol. 214, 211-214 (1999).

[0174] 4. Cogoni, C. & Macino, G. Gene silencing in Neurospora crassarequires a protein homologous to RNA-dependent RNA polymerase. Nature399, 166-169 (1999).

[0175] 5. Waterhouse, P. M., Graham, M. W. & Wang, M. B. Virusresistance and gene silencing in plants can be induced by simultaneousexpression of sense and antisense RNA. Proc. Natl Acad. Sci. USA 95,13959-13964 (1998).

[0176] 6. Montgomery, M. K. & Fire, A. Double-stranded RNA as a mediatorin sequence-specific genetic silencing and co-suppression. Trends Genet.14, 225-228 (1998).

[0177] 7. Ngo, H., Tschudi, C., Gull, K. & Ullu, E. Double-stranded RNAinduces mRNA degradation in Trypanosoma brucei. Proc. Natl Acad. Sci.USA 95, 14687-14692 (1998).

[0178] 8. Tabara, H. et al. The rde-1 gene, RNA interference, andtransposon silencing in C. elegans. Cell 99, 123-132 (1999).

[0179] 9. Ketting, R. F., Haverkamp, T. H. A., van Luenen, H. G. A. M. &Plasterk, R. H. A. mut-7 of C. elegans, required for transposonsilencing and RNA interference, is a homolog of Werner Syndrome helicaseand RnaseD. Cell 99, 133-141 (1999).

[0180] 10. Ratcliff, F., Harrison, B. D. & Baulcombe, D. C. A similaritybetween viral defense and gene silencing in plants. Science 276,1558-1560 (1997).

[0181] 11. Kennerdell, J. R. & Carthew, R. W. Use of dsRNA-mediatedgenetic interference to demonstrate that frizzled and frizzled 2 act inthe wingless pathway. Cell 95, 1017-1026 (1998).

[0182] 12. Misquitta, L. & Paterson, B. M. Targeted disruption of genefunction in Drosophila by RNA interference: a role for nautilus inembryonic somatic muscle formation. Proc. Natl Acad. Sci. USA 96,1451-1456 (1999).

[0183] 13. Kalejta, R. F., Brideau, A. D., Banfield, B. W. & Beavis, A.J. An integral membrane green fluorescent protein marker, Us9-GFP, isquantitatively retained in cells during propidium iodine-based cellcycle analysis by flow cytometry. Exp. Cell. Res. 248, 322-328 (1999).

[0184] 14. Wolf, D. A. & Jackson, P. K. Cell cycle: oiling the gears ofanaphase. Curr. Biol. 8, R637-R639 (1998).

[0185] 15. Kramer, E. R., Gieffers, C., Holz, G., Hengstschlager, M. &Peters, J. M. Activation of the human anaphase-promoting complex byproteins of the CDC20/fizzy family. Curr. Biol. 8, 1207-1210 (1998).

[0186] 16. Shuttleworth, J. & Colman, A. Antisenseoligonucleotide-directed cleavage of mRNA in Xenopus oocytes and eggs.EMBO J. 7, 427-434 (1988).

[0187] 17. Tabara, H., Grishok, A. & Mello, C. C. RNAi in C. elegans:soaking in the genome sequence. Science 282, 430-432 (1998).

[0188] 18. Bosher, J. M., Dufourcq, P., Sookhareea, S. & Labouesse, M.RNA interference can target pre-mRNA. Consequences for gene expressionin a Caenorhabditis elegans operon. Genetics 153, 1245-1256 (1999).

[0189] 19. Hamilton, J. A. & Baulcombe, D. C. A species of smallantisense RNA in posttranscriptional gene silencing in plants. Science286, 950-952 (1999).

[0190] 20. Jones, L. A., Thomas, C. L. & Maule, A. J. De novomethylation and co-suppression induced by a cytoplasmically replicatingplant RNA virus. EMBO J. 17, 6385-6393 (1998).

[0191] 21. Jones, L. A. et al. RNA-DNA interactions and DNA methylationin post-transcriptional gene silencing. Plant Cell 11, 2291-2301 (1999).

[0192] 22. Schneider, I. Cell lines derived from late embryonic stagesof Drosophila melanogaster. J. Embryol. Exp. Morpho. 27, 353-365 (1972).

[0193] 23. Di Nocera, P. P. & Dawid, I. B. Transient expression of genesintroduced into cultured cells of Drosophila. Proc. Natl Acad. Sci. USA80, 7095-7098 (1983).

Example 2 Role for a Bidentate Ribonuclease in the Initiation Step ofRNA Interference

[0194] Genetic approaches in worms, fungi and plants have identified agroup of proteins that are essential for double-stranded RNA-inducedgene silencing. Among these are ARGONAUTE family members (e.g. RDE1,QDE2)^(9,10,30), recQ-family helicases (MUT-7, QDE3)^(11,12), andRNA-dependent RNA polymerases (e.g. EGO-1, QDE1, SGS2/SDE1)¹³⁻¹⁶. Whilepotential roles have been proposed, none of these genes has beenassigned a definitive function in the silencing process. Biochemicalstudies have suggested that PTGS is accomplished by a multicomponentnuclease that targets mRNAs for degradation^(6,8,17). We have shown thatthe specificity of this complex may derive from the incorporation of asmall guide sequence that is homologous to the mRNA substrate⁶.Originally identified in plants that were actively silencingtransgenes⁷, these ˜22 nt. RNAs have been produced during RNAi in vitrousing an extract prepared from Drosophila embryos⁸. Putative guide RNAscan also be produced in extracts from Drosophila S2 cells (FIG. 5a).With the goal of understanding the mechanism of post-transcriptionalgene silencing, we have undertaken both biochemical fractionation andcandidate gene approaches to identify the enzymes that execute each stepof RNAi.

[0195] Our previous studies resulted in the partial purification of anuclease, RISC, that is an effector of RNA interference. See Example 1.This enzyme was isolated from Drosophila S2 cells in which RNAi had beeninitiated in vivo by transfection with dsRNA. We first sought todetermine whether the RISC enzyme and the enzyme that initiates RNAi viaprocessing of dsRNA into 22 mers are distinct activities. RISC activitycould be largely cleared from extracts by high-speed centrifugation(100,000×g for 60 min.) while the activity that produces 22 mersremained in the supernatant (FIG. 5b,c). This simple fractionationindicated that RISC and the 22 mer-generating activity are separable andthus distinct enzymes. However, it seems likely that they might interactat some point during the silencing process.

[0196] RNAse III family members are among the few nucleases that showspecificity for double-stranded RNA¹⁸. Analysis of the Drosophila and C.elegans genomes reveals several types of RNAse III enzymes. First is thecanonical RNAse III which contains a single RNAse III signature motifand a double-stranded RNA binding domain (dsRBD; e.g. RNC_CAEEL). Secondis a class represented by Drosha¹⁹, a Drosophila enzyme that containstwo RNAse III motifs and a dsRBD (CeDrosha in C. elegans). A third classcontains two RNAse III signatures and an amino terminal helicase domain(e.g. Drosophila CG4792, CG6493, C. elegans K12H4.8), and these hadpreviously been proposed by Bass as candidate RNAi nucleases²⁰.Representatives of all three classes were tested for the ability toproduce discrete, ˜22 nt. RNAs from dsRNA substrates.

[0197] Partial digestion of a 500 nt. cyclin E dsRNA with purified,bacterial RNAse III produced a smear of products while nearly completedigestion produced a heterogeneous group of ˜11-17 nucleotide RNAs (notshown). In order to test the dual-RNAse III enzymes, we prepared T7epitope-tagged versions of Drosha and CG4792. These were expressed intransfected S2 cells and isolated by immunoprecipitation usingantibody-agarose conjugates. Treatment of the dsRNA with the CG4792immunoprecipitate yielded ˜22 nt. fragments similar to those produced ineither S2 or embryo extracts (FIG. 6a). Neither activity in extract noractivity in immunoprecipitates depended on the sequence of the RNAsubstrate since dsRNAs derived from several genes were processedequivalently (see Supplement 1). Negative results were obtained withDrosha and with immunoprecipitates of a DExH box helicase (Homeless²¹;see FIGS. 6a,b). Western blotting confirmed that each of the taggedproteins was expressed and immunoprecipitated similarly (see Supplement2). Thus, we conclude that CG4792 may carry out the initiation step ofRNA interference by producing ˜22 nt. guide sequences from dsRNAs.Because of its ability to digest dsRNA into uniformly sized, small RNAs,we have named this enzyme Dicer (Dcr). Dicer mRNA is expressed inembryos, in S2 cells, and in adult flies, consistent with the presenceof functional RNAi machinery in all of these contexts (see Supplement3).

[0198] The possibility that Dicer might be the nuclease responsible forthe production of guide RNAs from dsRNAs prompted us to raise anantiserum directed against the carboxy-terminus of the Dicer protein(Dicer-1, CG4792). This antiserum could immunoprecipitate a nucleaseactivity from either Drosophila embryo extracts or from S2 cell lysatesthat produced ˜22 nt. RNAs from dsRNA substrates (FIG. 6C). The putativeguide RNAs that are produced by the Dicer-1 enzyme precisely comigratewith 22 mers that are produced in extract and with 22 mers that areassociated with the RISC enzyme (FIGS. 6 D,F). It had previously beenshown that the enzyme that produced guide RNAs in Drosophila embryoextracts was ATP-dependent⁸. Depletion of this cofactor resulted in an˜6-fold lower rate of dsRNA cleavage and in the production of RNAs witha slightly lower mobility. Of interest was the fact that both Dicer-1immunoprecipitates and extracts from S2 cells require ATP for theproduction of 22 mers (FIG. 6D). We do not observe the accumulation oflower mobility products in these cases, although we do routinely observethese in ATP-depleted embryo extracts. The requirement of this nucleasefor ATP is a quite unusual property. We hypothesize that thisrequirement could indicate that the enzyme may act processively on thedsRNA, with the helicase domain harnessing the energy of ATP hydrolysisboth for unwinding guide RNAs and for translocation along the substrate.

[0199] Efficient induction of RNA interference in C. elegans and inDrosophila has several requirements. For example, the initiating RNAmust be double-stranded, and it must be several hundred nucleotides inlength. To determine whether these requirements are dictated by Dicer,we characterized the ability of extracts and of immunoprecipitatedenzyme to digest various RNA substrates. Dicer was inactive againstsingle stranded RNAs regardless of length (see Supplement 4). The enzymecould digest both 200 and 500 nucleotide dsRNAs but was significantlyless active with shorter substrates (see Supplement 4). Double-strandedRNAs as short as 35 nucleotides could be cut by the enzyme, albeit veryinefficiently (data not shown). In contrast, E. coli RNAse III coulddigest to completion dsRNAs of 35 or 22 nucleotides (not shown). Thissuggests that the substrate preferences of the Dicer enzyme maycontribute to but not wholly determine the size dependence of RNAi.

[0200] To determine whether the Dicer enzyme indeed played a role inRNAi in vivo, we sought to deplete Dicer activity from S2 cells and testthe effect on dsRNA-induced gene silencing. Transfection of S2 cellswith a mixture of dsRNAs homologous to the two Drosophila Dicer genes(CG4792 and CG6493) resulted in an ˜6-7 fold reduction of Dicer activityeither in whole cell lysates or in Dicer-1 immunoprecipitates (FIGS.7A,B). Transfection with a control dsRNA (murine caspase 9) had noeffect. Qualitatively similar results were seen if Dicer was examined byNorthern blotting (not shown). Depletion of Dicer in this mannersubstantially compromised the ability of cells to silence subsequentlyan exogenous, GFP transgene by RNAi (FIG. 7C). These results indicatethat Dicer is involved in RNAi in vivo. The lack of complete inhibitionof silencing could result from an incomplete suppression of Dicer (whichis itself required for RNAi) or could indicate that in vivo, guide RNAscan be produced by more than one mechanism (e.g. through the action ofRNA-dependent RNA polymerases).

[0201] Our results indicate that the process of RNA interference can bedivided into at least two distinct steps. According to this model,initiation of PTGS would occur upon processing of a double-stranded RNAby Dicer into ˜22 nucleotide guide sequences, although we cannotformally exclude the possibility that another, Dicer-associated nucleasemay participate in this process. These guide RNAs would be incorporatedinto a distinct nuclease complex (RISC) that targets single-strandedmRNAs for degradation. An implication of this model is that guidesequences are themselves derived directly from the dsRNA that triggersthe response. In accord with this model, we have demonstrated that³²P-labeled, exogenous dsRNAs that have been introduced into S2 cells bytransfection are incorporated into the RISC enzyme as 22 mers (FIG. 7E).However, we cannot exclude the possibility that RNA-dependent RNApolymerases might amplify 22 mers once they have been generated orprovide an alternative method for producing guide RNAs.

[0202] The structure of the Dicer enzyme provokes speculation on themechanism by which the enzyme might produce discretely sized fragmentsirrespective of the sequence of the dsRNA (see Supplement 1, FIG. 8a).It has been established that bacterial RNAse III acts on its substrateas a dimer^(18,22,23). Similarly, a dimer of Dicer enzymes may berequired for cleavage of dsRNAs into ˜22 nt. pieces. According to onemodel, the cleavage interval would be determined by the physicalarrangement of the two RNAse III domains within Dicer enzyme (FIG. 8a).A plausible alternative model would dictate that cleavage was directedat a single position by the two RIII domains in a single Dicer protein.The 22 nucleotide interval could be dictated by interaction ofneighboring Dicer enzymes or by translocation along the mRNA substrate.The presence of an integral helicase domain suggests that the productsof Dicer cleavage might be single-stranded 22 mers that are incorporatedinto the RISC enzyme as such.

[0203] A notable feature of the Dicer family is its evolutionaryconservation. Homologs are found in C. elegans (K12H4.8), Arabidopsis(e.g., CARPEL FACTORY²⁴, T25K16.4, AC012328_(—)1), mammals(Helicase-MOI²⁵) and S. pombe (YC9A_SCHPO) (FIG. 8b, see Supplements 6,7for sequence comparisons). In fact, the human Dicer family member iscapable of generating ˜22 nt. RNAs from dsRNA substrates (Supplement 5)suggesting that these structurally similar proteins may all sharesimilar biochemical functions. It has been demonstrated that exogenousdsRNAs can affect gene function in early mouse embryos²⁹, and ourresults suggest that this regulation may be accomplished by anevolutionarily conserved RNAi machinery.

[0204] In addition to RNAseIII and helicase motifs, searches of the PFAMdatabase indicate that each Dicer family member also contains a ZAPdomain (FIG. 8c)²⁷. This sequence was defined based solely upon itsconservation in the Zwille/ARGONAUTE/Piwi family that has beenimplicated in RNAi by mutations in C. elegans (Rde-1)⁹ and Neurospora(Qde-2)¹⁰. Although the function of this domain is unknown, it isintriguing that this region of homology is restricted to two genefamilies that participate in dsRNA-dependent silencing. Both theARGONAUTE and Dicer families have also been implicated in commonbiological processes, namely the determination of stem-cell fates. Ahypomorphic allele of carpel factory, a member of the Dicer family inArabidopsis, is characterized by increased proliferation in floralmeristems²⁴. This phenotype and a number of other characteristicfeatures are also shared by Arabidopsis ARGONAUTE (ago1-1) mutants²⁶ (C.Kidner and R. Martiennsen, pers. comm.). These genetic analyses begin toprovide evidence that RNAi may be more than a defensive response tounusual RNAs but may also play important roles in the regulation ofendogenous genes.

[0205] With the identification of Dicer as a catalyst of the initiationstep of RNAi, we have begun to unravel the biochemical basis of thisunusual mechanism of gene regulation. It will be of critical importanceto determine whether the conserved family members from other organisms,particularly mammals, also play a role in dsRNA-mediated generegulation.

[0206] Methods

[0207] Plasmid constructs. A full-length cDNA encoding Drosha wasobtained by PCR from an EST sequenced by the Berkeley Drosophila genomeproject. The Homeless clone was a gift from Gillespie and Berg (Univ.Washington). The T7 epitope-tag was added to the amino terminus of eachby PCR, and the tagged cDNAs were cloned into pRIP, a retroviral vectordesigned specifically for expression in insect cells (E. Bernstein,unpublished). In this vector, expression is driven by the Orgyiapseudotsugata IE2 promoter (Invitrogen). Since no cDNA was available forCG4792/Dicer, a genomic clone was amplified from a bacmid (BACR23F10;obtained from the BACPAC Resource Center in the Dept. of Human Geneticsat the Roswell Park Cancer Institute). Again, during amplification, a T7epitope tag was added at the amino terminus of the coding sequence. Thehuman Dicer gene was isolated from a cDNA library prepared from HaCaTcells (GJH, unpublished). A T7-tagged version of the complete codingsequence was cloned into pCDNA3 (Invitrogen) for expression in humancells (LinX-A).

[0208] Cell culture and extract preparation. S2 and embryo culture. S2cells were cultured at 27° C. in 5% CO₂ in Schneider's insect mediasupplemented with 10% heat inactivated fetal bovine serum (Gemini) and1% antibiotic-antimycotic solution (Gibco BRL). Cells were harvested forextract preparation at 10×10⁶ cells/ml. The cells were washed 1× in PBSand were resuspended in a hypotonic buffer (10 mM Hepes pH 7.0, 2 mMMgCl2, 6 mM βME) and dounced. Cell lysates were spun 20,000×g for 20minutes. Extracts were stored at −80° C. Drosophila embryos were rearedin fly cages by standard methodologies and were collected every 12hours. The embryos were dechorionated in 50% chlorox bleach and washedthoroughly with distilled water. Lysis buffer (10 mM Hepes, 10 mM KCl,1.5 mM MgCl₂, 0.5 mM EGTA, 10 mM β-glycerophosphate, 1 mM DTT, 0.2 mMPMSF) was added to the embryos, and extracts were prepared byhomogenization in a tissue grinder. Lysates were spun for two hours at200,000×g and were frozen at −80° C. LinX-A cells, ahighly-transfectable derivative of human 293 cells, (Lin Xie and GJH,unpublished) were maintained in DMEM/10%FCS.

[0209] Transfections and immunoprecipitations. S2 cells were transfectedusing a calcium phosphate procedure essentially as previouslydescribed⁶. Transfection rates were ˜90% as monitored in controls usingan in situ β-galactosidase assay. LinX-A cells were also transfected bycalcium phosphate co-precipitation. For immunoprecipitations, cells(˜5×10⁶ per IP) were transfected with various clones and lysed threedays later in IP buffer (125 mM KOAc, 1 mM MgOAc, 1 mM CaCl₂, 5 mM EGTA,20 mM Hepes pH 7.0, 1 mM DTT, 1% NP-40 plus Complete protease inhibitors(Roche)). Lysates were spun for 10 minutes at 14,000×g and supernatantswere added to T7 antibody-agarose beads (Novagen). Antibody bindingproceeded for 4 hours at 4° C. Beads were centrifuged and washed inlysis buffer three times, and once in reaction buffer. The Dicerantiserum was raised in rabbits using a KLH-conjugated peptidecorresponding to the C-terminal 8 amino acids of Drosophila Dicer-1(CG4792).

[0210] Cleavage reactions. RNA preparation. Templates to be transcribedinto dsRNA were generated by PCR with forward and reverse primers, eachcontaining a T7 promoter sequence. RNAs were produced using Riboprobe(Promega) kits and were uniformly labeling during the transcriptionreaction with ³²P-UTP. Single-stranded RNAs were purified from 1%agarose gels. dsRNA cleavage. Five microliters of embryo or S2 extractswere incubated for one hour at 30° C. with dsRNA in a reactioncontaining 20 mM Hepes pH 7.0, 2 mM MgOAc, 2 mM DTT, 1 mM ATP and 5%Superasin (Ambion). Immunoprecipitates were treated similarly exceptthat a minimal volume of reaction buffer (including ATP and Superasin)and dsRNA were added to beads that had been washed in reaction buffer(see above). For ATP depletion, Drosophila embryo extracts wereincubated for 20 minutes at 30° C. with 2 mM glucose and 0.375 U ofhexokinase (Roche) prior to the addition of dsRNA.

[0211] Northern and Western analysis. Total RNA was prepared fromDrosophila embryos (0-12 hour), from adult flies, and from S2 cellsusing Trizol (Lifetech). Messenger RNA was isolated by affinityselection using magnetic oligo-dT beads (Dynal). RNAs wereelectrophoresed on denaturing formaldehyde/agarose gels, blotted andprobed with randomly primed DNAs corresponding to Dicer. For Westernanalysis, T7-tagged proteins were immunoprecipitated from whole celllysates in IP buffer using anti-T7-antibody-agarose conjugates. Proteinswere released from the beads by boiling in Laemmli buffer and wereseparated by electrophoresis on 8% SDS PAGE. Following transfer tonitrocellulose, proteins were visualized using an HRP-conjugated anti-T7antibody (Novagen) and chemiluminescent detection (Supersignal, Pierce).

[0212] RNAi of Dicer. Drosophila S2 cells were transfected either with adsRNA corresponding to mouse caspase 9 or with a mixture of two dsRNAscorresponding to Drosophila Dicer-1 and Dicer-2 (CG4792 and CG6493). Twodays after the initial transfection, cells were again transfected with amixture containing a GFP expression plasmid and either luciferase dsRNAor GFP dsRNA as previously described⁶. Cells were assayed for Diceractivity or fluorescence three days after the second transfection.Quantification of fluorescent cells was done on a Coulter EPICS cellsorter after fixation. Control transfections indicated that Diceractivity was not affected by the introduction of caspase 9 dsRNA.

References Cited Example 2

[0213] 1. Baulcombe, D. C. RNA as a target and an initiator ofpost-transcriptional gene silencing in transgenic plants. Plant Mol Biol32, 79-88 (1996).

[0214] 2. Wassenegger, M. & Pelissier, T. A model for RNA-mediated genesilencing in higher plants. Plant Mol Biol 37, 349-62 (1998).

[0215] 3. Montgomery, M. K. & Fire, A. Double-stranded RNA as a mediatorin sequence-specific genetic silencing and co-suppression [seecomments]. Trends Genet 14, 255-8 (1998).

[0216] 4. Sharp, P. A. RNAi and double-strand RNA. Genes Dev 13, 139-41(1999).

[0217] 5. Sijen, T. & Kooter, J. M. Post-transcriptional gene-silencing:RNAs on the attack or on the defense? [In Process Citation]. Bioessays22, 520-31 (2000).

[0218] 6. Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G. J. AnRNA-directed nuclease mediates post-transcriptional gene silencing inDrosophila cells. Nature 404, 293-6 (2000).

[0219] 7. Hamilton, A. J. & Baulcombe, D. C. A species of smallantisense RNA in posttranscriptional gene silencing in plants [seecomments]. Science 286, 950-2 (1999).

[0220] 8. Zamore, P. D., Tuschl, T., Sharp, P. A. & Bartel, D. P. RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to23 nucleotide intervals. Cell 101, 25-33 (2000).

[0221] 9. Tabara, H. et al. The rde-1 gene, RNA interference, andtransposon silencing in C. elegans. Cell 99, 123-32 (1999).

[0222] 10. Catalanotto, C., Azzalin, G., Macino, G. & Cogoni, C. Genesilencing in worms and fungi. Nature 404, 245 (2000).

[0223] 11. Ketting, R. F., Haverkamp, T. H., van Luenen, H. G. &Plasterk, R. H. Mut-7 of C. elegans, required for transposon silencingand RNA interference, is a homolog of Werner syndrome helicase andRNaseD. Cell 99, 133-41 (1999).

[0224] 12. Cogoni, C. & Macino, G. Posttranscriptional gene silencing inNeurospora by a RecQ DNA helicase. Science 286, 2342-4 (1999).

[0225] 13. Cogoni, C. & Macino, G. Gene silencing in Neurospora crassarequires a protein homologous to RNA-dependent RNA polymerase. Nature399, 166-9 (1999).

[0226] 14. Smardon, A. et al. EGO-1 is related to RNA-directed RNApolymerase and functions in germ-line development and RNA interferencein C. elegans [published erratum appears in Curr Biol May 18,2000;10(10):R393-4]. Curr Biol 10, 169-78 (2000).

[0227] 15. Mourrain, P. et al. Arabidopsis SGS2 and SGS3 genes arerequired for posttranscriptional gene silencing and natural virusresistance. Cell 101, 533-42 (2000).

[0228] 16. Dalmay, T., Hamilton, A., Rudd, S., Angell, S. & Baulcombe,D. C. An RNA-dependent RNA polymerase gene in Arabidopsis is requiredfor posttranscriptional gene silencing mediated by a transgene but notby a virus. Cell 101, 543-53 (2000).

[0229] 17. Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P. &Sharp, P. A. Targeted mRNA degradation by double-stranded RNA in vitro.Genes Dev 13, 3191-7 (1999).

[0230] 18. Nicholson, A. W. Function, mechanism and regulation ofbacterial ribonucleases. FEMS Microbiol Rev 23, 371-90 (1999).

[0231] 19. Filippov, V., Solovyev, V., Filippova, M. & Gill, S. S. Anovel type of RNase III family proteins in eukaryotes. Gene 245, 213-21(2000).

[0232] 20. Bass, B. L. Double-stranded RNA as a template for genesilencing. Cell 101, 235-8 (2000).

[0233] 21. Gillespie, D. E. & Berg, C. A. Homeless is required for RNAlocalization in Drosophila oogenesis and encodes a new member of theDE-H family of RNA-dependent ATPases. Genes Dev 9, 2495-508 (1995).

[0234] 22. Robertson, H. D., Webster, R. E. & Zinder, N. D. Purificationand properties of ribonuclease III from Escherichia coli. J Biol Chem243, 82-91 (1968).

[0235] 23. Dunn, J. J. RNase III cleavage of single-stranded RNA. Effectof ionic strength on the fideltiy of cleavage. J Biol Chem 251, 3807-14(1976).

[0236] 24. Jacobsen, S. E., Running, M. P. & Meyerowitz, E. M.Disruption of an RNA helicase/RNAse III gene in Arabidopsis causesunregulated cell division in floral meristems. Development 126, 5231-43(1999).

[0237] 25. Matsuda, S. et al. Molecular cloning and characterization ofa novel human gene (HERNA) which encodes a putative RNA-helicase.Biochim Biophys Acta 1490, 163-9 (2000).

[0238] 26. Bohmert, K. et al AGO1 defines a novel locus of Arabidopsiscontrolling leaf development. Embo J 17, 170-80 (1998).

[0239] 27. Sonnhammer, E. L., Eddy, S. R. & Durbin, R. Pfam: acomprehensive database of protein domain families based on seedalignments. Proteins 28, 405 -20 (1997).

[0240] 28. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs. Nucleic Acids Res 25,3389-402 (1997).

[0241] 29. Wianny, F. and Zernicka-Goetz, M. Specific interference withgene function by double-stranded RNA in early mouse development. NatureCell Biol. 2, 70-75 (2000).

[0242] 30. Fagard, M., Boutet, S., Morel, J.-B., Bellini, C. andVaucheret, H. Ago-1, Qde-2 and Rde-1 are related proteins required forpost-transcriptional gene silencing in plants, quelling in fungi, andRNA interference in animals. Proc. Natl. Acad. Sci. USA 97, 11650-11654(2000).

Example 3 A simplified Method for the Creation of Hairpin Constructs forRNA Interference

[0243] In numerous model organisms, double stranded RNAs have been shownto cause effective and specific suppression of gene function (ref. 1).This response, termed RNA interference or post-transcriptional genesilencing, has evolved into a highly effective reverse genetic tool inC. elegans, Drosophila, plants and numerous other systems. In thesecases, double-stranded RNAs can be introduced by injection, transfectionor feeding; however, in all cases, the response is both transient andsystemic. Recently, stable interference with gene expression has beenachieved by expression of RNAs that form snap-back or hairpin structures(refs 2-7). This has the potential not only to allow stable silencing ofgene expression but also inducible silencing as has been observed intrypanosomes and adult Drosophila (refs 2,4,5). The utility of thisapproach is somewhat hampered by the difficulties that arise in theconstruction of bacterial plasmids containing the long inverted repeatsthat are necessary to provoke silencing. In a recent report, it wasstated that more than 1,000 putative clones were screed to identify thedesired construct (ref 7).

[0244] The presence of hairpin structures often induces plasmidrearrangement, in part due to the E. coli sbc proteins that recognizeand cleave cruciform DNA structures (ref 8). We have developed a methodfor the construction of hairpins that does not require cloning ofinverted repeats, per se. Instead, the fragment of the gene that is tobe silenced is cloned as a direct repeat, and the inversion isaccomplished by treatment with a site-specific recombinase, either invitro (or potentially in vivo) (see FIG. 27). Following recombination,the inverted repeat structure is stable in a bacterial strain that lacksan intact SBC system (DL759). We have successfully used this strategy toconstruct numerous hairpin expression constructs that have beensuccessfully used to provoke gene silencing in Drosophila cells.

Literature Cited in Example 3

[0245] 1. Bosher, J. M. & Labouesse, M. RNA interference: genetic wandand genetic watchdog. Nat Cell Biol 2, E31-6 (2000).

[0246] 2. Fortier, E. & Belote, J. M. Temperature-dependent genesilencing by an expressed inverted repeat in Drosophila [publishederratum appears in Genesis; May 27, 2000; (1):47]. Genesis 26, 240-4(2000).

[0247] 3. Kennerdell, J. R. & Carthew, R. W. Heritable gene silencing inDrosophila using double-stranded RNA. Nat Biotechnol 18, 896-8 (2000).

[0248] 4. Lam, G. & Thummel, C. S. Inducible expression ofdouble-stranded RNA directs specific genetic interference in Drosophila[In Process Citation]. Curr Biol 10, 957-63 (2000).

[0249] 5. Shi, H. et al. Genetic interference in Trypanosoma brucei byheritable and inducible double-stranded RNA. Rna 6, 1069-76 (2000).

[0250] 6. Smith, N. A. et al Total silencing by intron-spliced hairpinRNAs. Nature 407, 319-20 (2000).

[0251] 7. Tavernarakis, N., Wang, S. L., Dorovkov, M., Ryazanov, A. &Driscoll, M. Heritable and inducible genetic interference bydouble-stranded RNA encoded by transgenes. Nat Genet 24, 180-3 (2000).

[0252] 8. Connelly, J. C. & Leach, D. R. The sbcC and sbcD genes ofEscherichia coli encode a nuclease involved in palindrome inviabilityand genetic recombination. Genes Cells 1, 285-91 (1996).

V. EQUIVALENTS

[0253] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

[0254] All of the above-cited references and publications are herebyincorporated by reference.

We claim:
 1. A method for attenuating expression of a target gene incultured cells, comprising introducing double stranded RNA (dsRNA) intothe cells in an amount sufficient to attenuate expression of the targetgene, wherein the dsRNA comprises a nucleotide sequence that hybridizesunder stringent conditions to a nucleotide sequence of the target gene.2. A method for attenuating expression of a target gene in a mammaliancell, comprising (i) activating one or both of a Dicer activity or anArgonaut activity in the cell, and (ii) introducing into the cell adouble stranded RNA (dsRNA) in an amount sufficient to attenuateexpression of the target gene, wherein the dsRNA comprises a nucleotidesequence that hybridizes under stringent conditions to a nucleotidesequence of the target gene.
 3. The method of claim 2, wherein the cellis suspended in culture.
 4. The method of claim 2, wherein the cell isin a whole animal, such as a non-human mammal.
 5. The method of claim 1or 2, wherein is engineered with (i) a recombinant gene encoding a Diceractivity, (ii) a recombinant gene encoding an Argonaut activity, or(iii) both.
 6. The method of claim 5, wherein the recombinant geneencodes a protein which includes an amino acid sequence at least 50percent identical to SEQ ID No. 2 or 4 or the Argonaut sequence shown inFIG.
 24. 7. The method of claim 5, wherein the recombinant gene includesa coding sequence hybridizes under wash conditions of 2×SSC at 22° C. toSEQ ID No. 1 or
 3. 8. The method of claim 1 or 2, wherein an endogenousDicer gene or Argonaut gene is activated.
 9. The method of claim 1 or 2,wherein the target gene is an endogenous gene of the cell.
 10. Themethod of claim 1 or 2, wherein the target gene is an heterologous generelative to the genome of the cell, such as a pathogen gene.
 11. Themethod of claim 1 or 2, wherein the cell is treated with an agent thatinhibits protein kinase RNA-activated (PKR) apoptosis, such as bytreatment with agents which inhibit expression of PKR, cause itsdestruction, and/or inhibit the kinase activity of PKF.
 12. The methodof claim 1 or 2, wherein the cell is a primate cell, such as a humancell.
 13. The method of claim 1 or 2, wherein the dsRNA is at least 20nucleotides in length.
 14. The method of claim 13, wherein the dsRNA isat least 100 nucleotides in length.
 15. The method of claim 1 or 2,wherein expression of the target gene is attenuated by at least 10 fold.16. An assay for identifying nucleic acid sequences responsible forconferring a particular phenotype in a cell, comprising (i) constructinga variegated library of nucleic acid sequences from a cell in anorientation relative to a promoter to produce double stranded DNA; (ii)introducing the variegated dsRNA library into a culture of target cells,which cells have an activated Dicer activity or Argonaut activity; (iii)identifying members of the library which confer a particular phenotypeon the cell, and identifying the sequence from a cell which correspond,such as being identical or homologous, to the library member.
 17. Amethod of conducting a drug discovery business comprising: (i)identifying, by the assay of claim 16, a target gene which provides aphenotypically desirable response when inhibited by RNAi; (ii)identifying agents by their ability to inhibit expression of the targetgene or the activity of an expression product of the target gene; (iii)conducting therapeutic profiling of agents identified in step (b), orfurther analogs thereof, for efficacy and toxicity in animals; and (iv)formulating a pharmaceutical preparation including one or more agentsidentified in step (iii) as having an acceptable therapeutic profile.18. The method of claim 17, including an additional step of establishinga distribution system for distributing the pharmaceutical preparationfor sale, and may optionally include establishing a sales group formarketing the pharmaceutical preparation.
 19. A method of conducting atarget discovery business comprising: (i) identifying, by the assay ofclaim 16, a target gene which provides a phenotypically desirableresponse when inhibited by RNAi; (ii) (optionally) conductingtherapeutic profiling of the target gene for efficacy and toxicity inanimals; and (iii). licensing, to a third party, the rights for furtherdrug development of inhibitors of the target gene.
 20. A method forattenuating expression of a target gene in a cell, comprisingintroducing into the cell a hairpin nucleic acid in an amount sufficientto attenuate expression of the target gene, wherein the hairpin nucleicacid comprises an inverted repeat of a nucleotide sequence thathybridizes under stringent conditions to a nucleotide sequence of thetarget gene.
 21. A hairpin nucleic acid for inhibiting expression of atarget gene, comprising a first nucleotide sequence that hybridizesunder stringent conditions to a nucleotide sequence of the target gene,and a second nucleotide sequence which is an complementary invertedrepeat of said first nucleotide sequence and hybridizes to said firstnucleotide sequence to form a hairpin structure.
 22. The method of claim20 or the hairpin nucleic acid of claim 21, wherein the hairpin nucleicis RNA.
 23. A non-human transgenic mammal having germline and/or somaticcells comprising a transgene encoding a dsRNA construct.
 24. Thetransgenic animal of claim 23, which is chimeric for said transgene. 25.The transgenic animal of claim 23, wherein said transgene ischromosomally incorporated.